Il-1 related polypeptides

ABSTRACT

The present invention is directed to novel polypeptides having homology to the IL-1-like family of proteins and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention, and methods for producing the polypeptides of the present invention.

RELATED APPLICATIONS

This is a continuation application of U.S. Ser. No. 09/869,566 filedFeb. 19, 2002, now pending, which is a National Phase application ofPCT/US99/30720 filed Dec. 22, 1999, now national, which claims thebenefit of U.S. Ser. No. 60/129,122 filed Apr. 13, 1999, U.S. Ser. No.60/116,843 filed Jan. 22, 1999, and U.S. Ser. No. 60/113,430 filed Dec.23, 1998, all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNAs having homology to interleukin-1 (IL-1) orinterleukin-1 receptor antagonist (IL-1Ra) polypeptides, and to therecombinant production of novel polypeptides, designated herein asinterleukin-1-like polypeptides (“IL-1lp”).

BACKGROUND OF THE INVENTION

Interleukin-1 refers to two proteins (IL-1α and IL-1β) which play a keyrole early in the inflammatory response (for a review, see Dinarello,Blood, 87: 2095-2147 (1996) and references therein). both proteins aremade as intracellular precursor proteins which are cleaved uponsecretion to yield mature carboxy-terminal 17 kDa fragments which arebiologically active. In the case of IL-1β, this cleavage involves anintracellular cysteine protease, known as ICE, which is required torelease the active fragment from the inactive precursor. The precursorof IL-1α is active.

These two proteins act by binding to cell surface receptors found onalmost all cell types and triggering a range of responses either aloneor in concert with other secreted factors. These range from effects onproliferation (e.g. fibroblasts, T cells) apoptosis (e.g. A375 melanomacells), cytokine induction (e.g. of TNF, IL-1, IL-8), receptoractivation (e.g. E-selectin), eicosanoid production (e.g. PGE2) and thesecretion of degradative enzymes (e.g. collagenase). To achieve theseeffects, IL-1 activates transcription factors such as NF-KB and AP-1.Several of the activities of IL-1 action on target cells are believed tobe mediated through activation of kinase cascades that have also beenassociated with cellular stresses, such as the stress activated MAPkinase JNK/SAPK and p38.

A third member of the IL-1 family was subsequently discovered which actsas a natural antagonist of IL-1α and IL-1β by binding to the IL-1receptor but not transducing an intracellular signal or a biologicalresponse. The protein is called IL-1Ra (for IL-1 receptor antagonist) orIRAP (for IL-1 receptor antagonist protein). At least threealternatively spliced forms of IL-1Ra exist: one encodes a secretedprotein, also known as secretory IL-1Ra (“sIL-1Ra”) (described inEisenberg et al., Nature, 343: 341-346 (1990)), and the other two encodeintracellular proteins. IL-1α, IL-1β and IL-1Ra exhibit approximately25-30% sequence identity with each other and share a similar threedimensional structure consisting of twelve β-strands folded into aβ-barrel, with an internal thrice repeated structural motif.

There are three known IL-1 receptor subunits. The active receptorcomplex consists of the type I receptor and IL-1 accessory protein(IL-1RAcP). The type I receptor is responsible for binding of the IL-1α,IL-1β and IL-1Ra ligands, and is able to do so in the absence of theIL-1RAcP. However, signal transduction requires the interaction of IL-1αor IL-1β with the IL-1RAcP. IL-1Ra does not interact with the IL-1RAcPand hence cannot induce signal transduction. A third receptor subunit,the type II receptor, binds IL-1α and IL-1β but cannot transduce signaldue its lack of an intracellular domain. Instead, the type II receptoreither acts as a decoy in its membrane bound form or as an IL-1antagonist in a processed, secreted form, and hence inhibits IL-1activity. The type II receptor weakly binds to IL-1Ra.

Many studies using IL-1Ra, soluble IL-1R derived from the extracellulardomain of the type I IL-1 receptor, antibodies to IL-1α or IL-1β, andtransgenic knockout mice for these genes have shown that IL-1 plays arole in a number of pathophysiologies (for a review, see Dinarello,Blood, 87: 2095-2147 (1996)). For example, IL-1Ra has been shown to beeffective in animal models of septic shock, rheumatoid arthritis,graft-versus-host disease (GVHD), stroke, cardiac ischemia, psoriasis,inflammatory bowel disease, and asthma. In addition, IL-1Ra hasdemonstrated efficacy in clinical trials for rheumatoid arthritis andGVHD, and is also in clinical trials for inflammatory bowel disease,asthma and psoriasis.

More recently, interleukin-18 (IL-18) was placed in the IL-1 family (fora review, see Dinarello et al, J. Leukocyte Biol., 63: 658-664 (1998)).IL-18 shares the β-pleated, barrel-like form of IL-1α and IL-1β. Inaddition, IL-18 is the natural ligand for the IL-1 receptor familymember formerly known as IL-1R-related protein (IL-1Rrp) (now known asthe IL-18 receptor (IL-18R)). IL-18 has been shown to initiate theinflammatory cytokine cascade in a mixed population of peripheral bloodmononuclear cells (PBMCs) by triggering the constitutive IL-18 receptorson lymphocytes and NK cells, inducing TNF production in the activatedcells. TNF, in turn, stimulates IL-1 and IL-8 production in CD14+ cells.Because of its ability to induce TNF, IL-1, and both C—C and C—X—Cchemokines, and because IL-18 induces Fas ligand as well as nucleartranslocation of nuclear factor κB (NF-κB), IL-18 ranks with otherpro-inflammatory cytokines as a likely contributor to systemic and localinflammation.

SUMMARY OF THE INVENTION

A family of cDNA clones (DNA85066, DNA96786, DNA94618, DNA102043,DNA114876, DNA102044, DNA92929, DNA96787, and DNA92505) has beenidentified, having homology to interleukin-1, that encode novelpolypeptides. The novel polypeptides and variants thereof arecollectively designated in the present application as“interleukin-1-like polypeptides” or “IL-1lp”, as further definedherein. Accordingly, one aspect of the invention is an isolated IL-1lppolypeptide.

In another embodiment, the invention provides an isolated nucleic acidmolecule encoding an IL-1lp polypeptide.

In another embodiment, the invention provides a method for producing anIL-1lp comprising culturing a host cell comprising a heterologousnucleic acid sequence encoding an IL-1lp polypeptide, under conditionswherein the IL-1lp polypeptide is expressed, and recovering the IL-1lppolypeptide from the host cell.

In another embodiment, the invention provides an anti-IL-1lp antibody.

In another embodiment, the invention provides chimeric moleculescomprising an IL-1lp polypeptide fused to a heterologous polypeptide oramino acid sequence. An example of such a chimeric molecule comprises anIL-1lp polypeptide fused to an epitope tag sequence or a Fc region of animmunoglobulin.

In another embodiment, the invention provides an antibody whichspecifically binds to an IL-1lp polypeptide. Optionally, the antibody isa monoclonal antibody.

In yet another embodiment, the invention concerns agonists andantagonists of a native IL-1lp polypeptide. In a particular embodiment,the agonist or antagonist is an anti-IL-1lp antibody.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists of a native IL-1lp polypeptide, by contactingthe native IL-1lp polypeptide with a candidate molecule and monitoring abiological activity mediated by said polypeptide.

In a still further embodiment, the invention concerns a compositioncomprising an IL-1lp polypeptide, or an agonist or antagonist ashereinabove defined, in combination with a pharmaceutically acceptablecarrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) and derived amino acidsequences (SEQ ID NOS:2-3) related to a native sequence hIL-1Ra1. Thenucleotide sequence (SEQ ID NO:1) contains an intron believed to extendfrom nucleotide positions 181 to 432, with a splice donor site atnucleotide positions 181 to 186 and splice acceptor site at nucleotidepositions 430 to 432. The amino acid sequences (SEQ ID NOS:2 and 3) arederived from the exonic sequences that are believed to make up theprocessed (intron-free) coding sequence.

FIG. 2 shows the nucleotide sequence (SEQ ID NO:4) and derived aminoacid sequence (SEQ ID NO:5) of a native sequence hIL-1Ra1 polypeptidefused at its N-terminus to a heterologous signal peptide (amino acidpositions 1-15), flag peptide affinity handle (amino acid positions16-23) and peptide linker (amino acid positions 24-36).

FIG. 3 shows the nucleotide sequence (SEQ ID NO:6) and derived aminoacid sequence (SEQ ID NO:7) of a native sequence hIL-1Ra1 polypeptide.The nucleotide sequence (SEQ ID NO:6) and derived amino acid sequence(SEQ ID NO:7) are believed to represent the processed (intron-free) formand intact hIL-1Ra1 polypeptide, respectively, of the nucleotidesequence (SEQ ID NO:1) and amino acid sequences (SEQ ID NOS:2-3) ofFIG. 1. The start and stop codons in the coding sequence are located atnucleotide positions 103-105 and 682-684, respectively. The putativesignal sequence extends from amino acid positions 1 to 14. A putativecAMP- and cGMP-dependent protein kinase phosphorylation site is locatedat amino acid positions 33-36. Putative N-myristoylation sites arelocated at amino acid positions 50-55 and 87-92.

FIG. 4 shows the nucleotide sequence (SEQ ID NO:8) of EST AI014548.

FIG. 5 shows the nucleotide sequence (SEQ ID NO:9) and derived aminoacid sequence (SEQ ID NO:10) of a native sequence hIL-1Ra2 polypeptide.The start and stop codons in the coding sequence are located atnucleotide positions 96-98 and 498-500, respectively. The putativesignal sequence extends from amino acid positions 1-26.

FIG. 6 shows the nucleotide sequence (SEQ ID NO:11) of EST 1433156.

FIG. 7 shows the nucleotide sequence (SEQ ID NO:12) and derived aminoacid sequence (SEQ ID NO:13) of a native sequence hIL-1Ra3 polypeptide.The start and stop codons in the coding sequence are located atnucleotide positions 1-3 and 466-468, respectively. The putative signalsequence extends from amino acid positions 1-33. PutativeN-myristoylation sites are located at amino acid positions 29-34, 30-35,60-65, 63-68, 73-78, 91-96 and 106-111. An interleukin-1-like sequenceis located at amino acid positions 111-131.

FIG. 8 shows the nucleotide sequence (SEQ ID NO:14) of EST 5120028.

FIG. 9 shows the nucleotide sequence (SEQ ID NO:15) and derived aminoacid sequence (SEQ ID NO:16) of a native sequence mIL-1Ra3 polypeptide.The start and stop codons in the coding sequence are located atnucleotide positions 145-147 and 610-612, respectively. The putativesignal sequence extends from amino acid positions 1-33. PutativeN-myristoylation sites are located at amino acid positions 29-34, 60-65,63-68, 91-96 and 106-111. An interleukin-1-like sequence is located atamino acid positions 111-131.

FIG. 10 shows the nucleotide sequence (SEQ ID NO:17) of EST W08205.

FIG. 11 is an autoradiograph of Northern blots depicting expression ofhIL-1Ra3 mRNA in placental tissue and expression of mIL-1Ra3 mRNA inday-17 mouse embryo tissue.

FIG. 12 is an amino acid sequence alignment of native sequence hIL-1Ra1L(SEQ ID NO:19), hIL-1Ra1V (SEQ ID NO:25), hIL-1Ra1S (SEQ ID NO:21),hIL-1Ra2 (SEQ ID NO:10), hIL-1Ra3 (SEQ ID NO:13) and mIL-1Ra3 (SEQ IDNO:16) polypeptides with secretory hIL-1Ra (also referred to as“sIL-1Ra” and “hIL-1Ra”) (SEQ ID NO:26), hIL-1Raβ (SEQ ID NO:27) andTANGO-77 (SEQ ID NO:28).

FIG. 13A is a Western blot depicting the interleukin-18 receptor(IL-18R) binding activity of hIL-1Ra1. In the top panel (depicting aprotein band at approximately 22 kD), a conditioned medium containingFLAGhIL-1Ra1 and FLAGIL-1R-ECD-Fc (shown in the left lane) and aconditioned medium containing FLAGhIL-1Ra1 and FLAGIL-18R-ECD-Fc (shownin the right lane) were each immunoprecipitated with proteinG-sepharose, and the resulting precipitates were resolved by gelelectrophoresis and Western blotting with anti-FLAG monoclonal antibody.In the middle and bottom panels (depicting protein bands atapproximately 22 kD and 85 kD), a second aliquot from the FLAGhIL-1Ra1and FLAGIL-1R-ECD-Fc conditioned medium used in the top panel (shown inthe left lane) and a second aliquot from the FLAGhIL-1Ra1 andFLAGIL-18R-ECD-Fc conditioned medium used in the top panel (shown in theright lane) were each immunoprecipitated with anti-FLAG monoclonalantibody, and the resulting precipitates were resolved by gelelectrophoresis and Western blotting with anti-FLAG monoclonal antibody.

FIG. 13B is a Western blot depicting the IL-1R binding activity ofhIL-1Ra3. In the top panel (depicting a protein band at approximately 20kD), a conditioned medium containing hIL-1Ra3-FLAG and FLAGDR6-Fc (shownin the left lane), a conditioned medium containing hIL-1Ra3-FLAG andFLAGIL-1R-ECD-Fc (shown in the middle lane), and conditioned mediumcontaining hIL-1Ra3-FLAG and FLAGIL-18R-ECD-Fc (shown in the right lane)were each immunoprecipitated with protein G sepharose, and the resultingprecipitates were resolved by gel electrophoresis and Western blottingwith anti-FLAG monoclonal antibody. In the middle and bottom panels(depicting protein bands at approximately 20 kD and 85 kD), a secondaliquot from the hIL-1Ra3-FLAG and FLAGDR6-Fc conditioned medium used inthe top panel (shown in the left lane), a second aliquot from thehIL-1Ra3-FLAG and FLAGIL-1R-ECD-Fc conditioned medium used in the toppanel (shown in the middle lane) and a second aliquot from thehIL-1Ra3-FLAG and FLAGIL-18R-ECD-Fc conditioned medium used in the toppanel (shown in the right lane) were each immunoprecipitated withanti-FLAG monoclonal antibody, and the resulting precipitates wereresolved by gel electrophoresis and Western blotting with anti-FLAGmonoclonal antibody.

FIG. 14 is a Western blot depicting the interleukin-1 receptor (IL-1R)binding activity of mIL-1Ra3. In the top panel (depicting a protein bandat approximately 21 kD) and the bottom panel (depicting protein bands atapproximately 85 kD) the FLAGIL-1R-ECD-Fc in conditioned medium (shownin the left lane) and the FLAGIL-18R-ECD-Fc in conditioned medium (shownin the right lane) were immobilized with protein G-agarose, theresulting solid phase was contacted with conditioned medium containingFLAGmIL-1Ra3, and the resulting bound complexes were resolved by gelelectrophoresis and Western blotting with anti-FLAG monoclonal antibody.

FIG. 15 shows the nucleotide sequence (SEQ ID NO:18) and derived aminoacid sequence (SEQ ID NO:19) of a native sequence hIL-1Ra1L polypeptide.The start and stop codons in the coding sequence are located atnucleotide positions 4-6 and 625-627, respectively. The putative signalsequence extends from amino acid positions 1 to 34. A putative cAMP- andcGMP-dependent protein kinase phosphorylation site is located at aminoacid positions 47-50. Putative N-myristoylation sites are located atamino acid positions 64-69 and 101-106.

FIG. 16 shows the nucleotide sequence (SEQ ID NO:20) and derived aminoacid sequence (SEQ ID NO:21) of a native sequence hIL-1Ra1S polypeptide.The start and stop codons in the coding sequence are located atnucleotide positions 4-6 and 505-507, respectively. A putative signalsequence extends from amino acid positions 1 to 46. A putativeN-myristoylation site is located at amino acid positions 61-66.

FIG. 17 shows the single stranded nucleotide sequence (SEQ ID NO:23) ofEST AI343258 (lower strand) along with its complementary nucleotidesequence (SEQ ID NO:22) (upper strand).

FIG. 18 is an amino acid sequence alignment of native sequence hIL-1Ra1(SEQ ID NO:3), hIL-1Ra1L (SEQ ID NO:19), hIL-1Ra1V (SEQ ID NO:25) andhIL-1Ra1S (SEQ ID NO:21) polypeptides.

FIG. 19 shows the nucleotide sequence (SEQ ID NO:24) and derived aminoacid sequence (SEQ ID NO:25) of a native sequence hIL-1Ra1V polypeptide.The start and stop codons in the coding sequence are located atnucleotide positions 73-75 and 727-729, respectively. An alternate startcodon is located at nucleotide positions 106-108. A putative signalsequence extends from amino acid positions 1 to 48.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions:

The terms “interleukin-1-like polypeptide”, “interleukin-1-likeprotein”, “IL-1lp”, “IL-1lp polypeptide”, and “IL-1lp protein” encompassany native sequence IL-1lp, and further encompass IL-1lp variants (whichare further defined herein). The IL-1lp may be isolated from a varietyof sources, such as from human tissue types or from another source, orprepared by recombinant and/or synthetic methods.

A “native sequence IL-1lp” comprises a polypeptide having the same aminoacid sequence as a native sequence hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V,hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, or mIL-1Ra3, (which are further definedherein). Such native sequence IL-1lp can be isolated from nature or canbe produced by recombinant and/or synthetic means. The term “nativesequence IL-1lp” specifically encompasses naturally-occurring truncatedor secreted forms (e.g., a processed, mature sequence) andnaturally-occurring allelic variants of the IL-1lp.

The terms “naturally-occurring amino acid sequence” and “native aminoacid sequence” mean any amino acid sequence found in a polypeptideexisting in nature, i.e. present in a naturally-occurring polypeptide.

The terms “non-naturally-occurring amino acid sequence” and “non-nativeamino acid sequence” mean any amino acid sequence not found in apolypeptide existing in nature, i.e. not present in anaturally-occurring polypeptide.

“IL-1lp variant” is defined as any polypeptide that comprises a variantof hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, ormIL-1Ra3 (which are further defined herein).

Human interleukin-1 receptor antagonist analog 1 (“hIL-1Ra1”), hIL-1Ra1polypeptide, and hIL-1Ra1 protein are defined as any native sequencehIL-1Ra1 or variant hIL-1Ra1.

A “native sequence hIL-1Ra1” means a polypeptide comprising anaturally-occurring amino acid sequence selected from the groupconsisting of: (1) the amino acid sequence of amino acid residues fromat or about 37 to at or about 63 of FIG. 2 (SEQ ID NO:5); (2) the aminoacid sequence of amino acid residues from at or about 37 to at or about203 of FIG. 2 (SEQ ID NO:5); (3) the amino acid sequence of amino acidresidues from at or about 15 to about 53 of FIG. 3 (SEQ ID NO:7); (4)the amino acid sequence of amino acid residues from at or about 15 to ator about 193 of FIG. 3 (SEQ ID NO:7); and (5) the amino acid sequence ofany naturally-occurring truncated or secreted form or anynaturally-occurring allelic variant of a polypeptide comprising theamino acid sequence of (1) or (2) or (3) or (4). In one embodiment ofthe invention, the native sequence hIL-1Ra1 comprises amino acids fromat or about 37 to at or about 203 of FIG. 2 (SEQ ID NO:5) or amino acidsfrom at or about 15 to at or about 193 of FIG. 3 (SEQ ID NO:7).

“hIL-1Ra1 variant” is defined as any hIL-1Ra1 N-terminal variant orhIL-1Ra1 full sequence variant (which are further defined herein).

“hIL-1Ra1 N-terminal variant” means any hIL-1Ra1 other than a nativesequence hIL-1Ra1, which variant is an active hIL-1Ra1, as definedbelow, having at least about 80% amino acid sequence identity with anamino acid sequence selected from the group consisting of: (1) the aminoacid sequence of amino acid residues from at or about 37 to at or about63 of FIG. 2 (SEQ ID NO:5); and (2) the amino acid sequence of aminoacid residues from at or about 15 to at or about 53 of FIG. 3 (SEQ IDNO:7). Such hIL-1Ra1 N-terminal variants include, for instance, hIL-1Ra1polypeptides wherein one or more amino acid residues are added, ordeleted, internally or at the N- or C-terminus, in the sequence of aminoacid residues from at or about 37 to at or about 63 of FIG. 2 (SEQ IDNO:5) or in the sequence of amino acid residues from at or about 15 toat or about 53 of FIG. 3 (SEQ ID NO:7). Ordinarily, an hIL-1Ra1N-terminal variant will have at least about 80% amino acid sequenceidentity, or at least about 85% amino acid sequence identity, or atleast about 90% amino acid sequence identity, or at least about 95%amino acid sequence identity with an amino acid sequence selected fromthe group consisting of: (1) the amino acid sequence of amino acidresidues from at or about 37 to at or about 63 of FIG. 2 (SEQ ID NO:5);and (2) the amino acid sequence of amino acid residues from at or about15 to at or about 53 of FIG. 3 (SEQ ID NO:7).

“hIL-1Ra1 full sequence variant” means any hIL-1Ra1 other than a nativesequence hIL-1Ra1, which variant retains at least one biologic activityof a native sequence hIL-1Ra1, such as the ability to bind IL-18R, andwhich variant has at least about 80% amino acid sequence identity, or atleast about 85% amino acid sequence identity, or at least about 90%amino acid sequence identity, or at least about 95% amino acid sequenceidentity with an amino acid sequence selected from the group consistingof: (1) the amino acid sequence of amino acid residues from at or about37 to at or about 203 of FIG. 2 (SEQ ID NO:5); and (2) the amino acidsequence of amino acid residues from at or about 15 to at or about 193of FIG. 3 (SEQ ID NO:7). Such hIL-1Ra1 full sequence variants include,for instance, hIL-1Ra1 polypeptides wherein one or more amino acidresidues are added, or deleted, internally or at the N- or C-terminus,in the sequence of amino acid residues from at or about 37 to at orabout 203 of FIG. 2 (SEQ ID NO:5) or in the sequence of amino acidresidues from at or about 15 to at or about 193 of FIG. 3 (SEQ ID NO:7).

Human interleukin-1 receptor antagonist analog 1 long (“hIL-1Ra1L”),hIL-1Ra1L polypeptide, and hIL-1Ra1L protein are defined as any nativesequence hIL-1Ra1L or hIL-1Ra1L variant (which are further definedherein).

A “native sequence hIL-1Ra1L” means a polypeptide comprising anaturally-occurring amino acid sequence selected from the groupconsisting of: (1) the amino acid sequence of amino acid residues fromat or about 26 to at or about 44 of FIG. 15 (SEQ ID NO:19); (2) theamino acid sequence of amino acid residues from at or about 26 to at orabout 207 of FIG. 15 (SEQ ID NO:19); and (3) the amino acid sequence ofany naturally-occurring truncated or secreted form or anynaturally-occurring allelic variant of a polypeptide comprising theamino acid sequence of (1) or (2). In one embodiment of the invention,the native sequence hIL-1Ra1L comprises amino acids from at or about 26to at or about 207 of FIG. 15 (SEQ ID NO:19).

“hIL-1Ra1L variant” is defined as any hIL-1Ra1L N-terminal variant orhIL-1Ra1L full sequence variant or hIL-1Ra1L fusion variant (which arefurther defined herein).

“hIL-1Ra1L N-terminal variant” means any hIL-1Ra1L other than a nativesequence hIL-1Ra1L, which variant is an active hIL-1Ra1L, as definedbelow, having at least about 80% amino acid sequence identity with theamino acid sequence of amino acid residues from at or about 26 to at orabout 44 of FIG. 15 (SEQ ID NO:19). Such hIL-1Ra1L N-terminal variantsinclude, for instance, hIL-1Ra1L polypeptides wherein one or more aminoacid residues are added, or deleted, internally or at the N- orC-terminus, in the sequence of amino acid residues from at or about 26to at or about 44 of FIG. 15 (SEQ ID NO:19). Ordinarily, an hIL-1Ra1LN-terminal variant will have at least about 80% amino acid sequenceidentity, or at least about 85% amino acid sequence identity, or atleast about 90% amino acid sequence identity, or at least about 95%amino acid sequence identity with the amino acid sequence of amino acidresidues from at or about 26 to at or about 44 of FIG. 15 (SEQ IDNO:19).

“hIL-1Ra1L full sequence variant” means any hIL-1Ra1L other than anative sequence hIL-1Ra1L, which variant retains at least one biologicactivity of a native sequence hIL-1Ra1L, such as the ability to bindIL-18R, and which variant has at least about 80% amino acid sequenceidentity, or at least about 85% amino acid sequence identity, or atleast about 90% amino acid sequence identity, or at least about 95%amino acid sequence identity with the amino acid sequence of amino acidresidues from at or about 26 to at or about 207 of FIG. 15 (SEQ IDNO:19). Such hIL-1Ra1L full sequence variants include, for instance,hIL-1Ra1L polypeptides wherein one or more amino acid residues areadded, or deleted, internally or at the N- or C-terminus, in thesequence of amino acid residues from at or about 26 to at or about 207of FIG. 15 (SEQ ID NO:19).

“hIL-1Ra1L fusion variant” means a chimeric hIL-1Ra1L consisting of anative sequence hIL-1Ra1L fused at its N- or C-terminus to aheterologous amino acid or amino acid sequence. In one embodiment, thehIL-1Ra1L fusion variant polypeptide consists of a native sequence ofhIL-1Ra1L fused at its N-terminus or C-terminus to a heterologous aminoacid or amino acid sequence, wherein the heterologous amino acid oramino acid sequence is heterologous to the native sequence, i.e. theresulting chimeric sequence is non-naturally occurring. In anotherembodiment, the hIL-1Ra1L fusion variant consists of the amino acidsequence of amino acids from at or about 26 to at or about 207,inclusive of FIG. 15 (SEQ ID NO:19), or the amino acid sequence of aminoacid residues from at or about 1 to at or about 207, inclusive of FIG.15 (SEQ ID NO:19), fused at its N-terminus or C-terminus to aheterologous amino acid or amino acid sequence to form a non-naturallyoccurring fusion protein. Such hIL-1Ra1L fusion variants include, forinstance, hIL-1Ra1L polypeptides wherein a heterologous secretion leadersequence is fused to the N-terminus of the sequence of amino acidresidues from at or about 26 to at or about 207 of FIG. 15 (SEQ IDNO:19), or amino acid residues from at or about 1 to at or about 207 ofFIG. 15 (SEQ ID NO:19).

Human interleukin-1 receptor antagonist analog 1 long allelic variant(“hIL-1Ra1V”), hIL-1Ra1V polypeptide, and hIL-1Ra1V protein are definedas any native sequence hIL-1Ra1V or hIL-1Ra1V variant (which are furtherdefined herein).

A “native sequence hIL-1Ra1V” means a polypeptide comprising anaturally-occurring amino acid sequence selected from the groupconsisting of: (1) the amino acid sequence of amino acid residues fromat or about 46 to at or about 55 of FIG. 19 (SEQ ID NO:25); (2) theamino acid sequence of amino acid residues from at or about 46 to at orabout 218 of FIG. 19 (SEQ ID NO:25); (3) the amino acid sequence ofamino acid residues from at or about 37 to at or about 218 of FIG. 19(SEQ ID NO:25); (4) the amino acid sequence of amino acid residues fromat or about 12 to at or about 218 of FIG. 19 (SEQ ID NO:25); and (5) theamino acid sequence of any naturally-occurring truncated or secretedform or any naturally-occurring allelic variant of a polypeptidecomprising the amino acid sequence of (1) or (2) or (3) or (4). In oneembodiment of the invention, the native sequence hIL-1Ra1V comprisesamino acids from at or about 46 to at or about 218 of FIG. 19 (SEQ IDNO:25), or amino acids from at or about 37 to at or about 218 of FIG. 19(SEQ ID NO:25), or amino acids from at or about 12 to at or about 218 ofFIG. 19 (SEQ ID NO:25), or amino acids from at or about 1 to at or about218 of FIG. 19 (SEQ ID NO:25).

“hIL-1Ra1V variant” is defined as any hIL-1Ra1V N-terminal variant orhIL-1Ra1V full sequence variant or hIL-1Ra1V fusion variant (which arefurther defined herein).

“hIL-1Ra1V N-terminal variant” is defined as any hIL-1Ra1V other than anative sequence hIL-1Ra1V, which variant is an active hIL-1Ra1V, asdefined below, having at least about 80% amino acid sequence identitywith the amino acid sequence of amino acid residues from at or about 46to at or about 89 of FIG. 19 (SEQ ID NO:25). Such hIL-1Ra1V N-terminalvariants include, for instance, hIL-1Ra1V polypeptides wherein one ormore amino acid residues are added, internally or at the N- orC-terminus, in the sequence of amino acid residues from at or about 46to at or about 89 of FIG. 19 (SEQ ID NO:25). Ordinarily, an hIL-1Ra1VN-terminal variant will have at least about 80% amino acid sequenceidentity, or at least about 85% amino acid sequence identity, or atleast about 90% amino acid sequence identity, or at least about 95%amino acid sequence identity with the sequence of amino acid residuesfrom at or about 46 to at or about 89 of FIG. 19 (SEQ ID NO:25).

“hIL-1Ra1V full sequence variant” means any hIL-1Ra1V other than anative sequence hIL-1Ra1V, which variant retains at least one biologicactivity of a native sequence hIL-1Ra1V, such as the ability to bindIL-118R, and which variant has at least about 80% amino acid sequenceidentity, or at least about 85% amino acid sequence identity, or atleast about 90% amino acid sequence identity, or at least about 95%amino acid sequence identity with the sequence of amino acid residuesfrom at or about 46 to at or about 218 of FIG. 19 (SEQ ID NO:25).

“hIL-1Ra1V fusion variant” means a chimeric hIL-1Ra1V consisting of anative sequence hIL-1Ra1V fused at its N- or C-terminus to aheterologous amino acid or amino acid sequence. In one embodiment, thehIL-1Ra1V fusion variant polypeptide consists of a native sequence ofhIL-1Ra1V fused at its N-terminus or C-terminus to a heterologous aminoacid or amino acid sequence, wherein the heterologous amino acid oramino acid sequence is heterologous to the native sequence, i.e. theresulting chimeric sequence is non-naturally occurring. In anotherembodiment, the hIL-1Ra1V fusion variant consists of the amino acidsequence of amino acids from at or about 46 to at or about 218 of FIG.19 (SEQ ID NO:25), or the amino acid sequence of amino acids from at orabout 37 to at or about 218 of FIG. 19 (SEQ ID NO:25), or the amino acidsequence of amino acids from at or about 12 to at or about 218 of FIG.19 (SEQ ID NO:25), or the amino acid sequence of amino acids from at orabout 1 to at or about 218 of FIG. 19 (SEQ ID NO:25), fused at itsN-terminus or C-terminus to a heterologous amino acid sequence to form anon-naturally occurring fusion protein. Such hIL-1Ra1V fusion variantsinclude, for instance, hIL-1Ra1V polypeptides wherein a heterologoussecretion leader sequence is fused to the N-terminus of the sequence ofamino acid residues from at or about 46 to at or about 218 of FIG. 19(SEQ ID NO:25), or amino acid residues from at or about 37 to at orabout 218 of FIG. 19 (SEQ ID NO:25), or amino acid residues from at orabout 12 to at or about 218 of FIG. 19 (SEQ ID NO:25), or amino acidresidues from at or about 1 to at or about 218 of FIG. 19 (SEQ IDNO:25).

Human interleukin-1 receptor antagonist analog 1 short (“hIL-1Ra1S”),hIL-1Ra1S polypeptide, and hIL-1Ra1S protein are defined as any nativesequence hIL-1Ra1S or hIL-1Ra1S variant (which are further definedherein).

A “native sequence hIL-1Ra1S” means a polypeptide comprising anaturally-occurring amino acid sequence selected from the groupconsisting of: (1) the amino acid sequence of amino acid residues fromat or about 1 to at or about 38 of FIG. 16 (SEQ ID NO:21); (2) the aminoacid sequence of amino acid residues from at or about 26 to at or about167 of FIG. 16 (SEQ ID NO:21); (3) the amino acid sequence of amino acidresidues from at or about 39 to at or about 167 of FIG. 16 (SEQ IDNO:21); (4) the amino acid sequence of amino acid residues from at orabout 47 to at or about 167 of FIG. 16 (SEQ ID NO:21); and (5) the aminoacid sequence of any naturally-occurring truncated or secreted form orany naturally-occurring allelic variant of a polypeptide comprising theamino acid sequence of (1) or (2) or (3) or (4). In one embodiment ofthe invention, the native sequence hIL-1Ra1S comprises amino acids fromat or about 26 to at or about 167 of FIG. 16 (SEQ ID NO:21), or aminoacids from at or about 1 to at or about 167 of FIG. 16 (SEQ ID NO:21).In another embodiment, the native sequence hIL-1Ra1S consists of aminoacids from at or about 47 to at or about 167 of FIG. 16 (SEQ ID NO:21)or amino acids from at or about 39 to at or about 167 of FIG. 16 (SEQ IDNO:21).

“hIL-1Ra1S fusion variant” and “hIL-1Ra1S variant” mean a chimerichIL-1Ra1S consisting of a native sequence hIL-1Ra1S fused at itsN-terminus or C-terminus to a heterologous amino acid or amino acidsequence. In one embodiment, the hIL-1Ra1S fusion variant polypeptideconsists of a native sequence of hIL-1Ra1S fused at its N-terminus orC-terminus to a heterologous amino acid or amino acid sequence, whereinthe heterologous amino acid or amino acid sequence is heterologous tothe native sequence, i.e. the resulting chimeric sequence isnon-naturally occurring. In another embodiment, the hIL-1Ra1S fusionvariant consists of the amino acid sequence of amino acids from at orabout 47 to at or about 167 of FIG. 16 (SEQ ID NO:21), or the amino acidsequence of amino acids from at or about 39 to at or about 167 of FIG.16 (SEQ ID NO:21), fused at its N-terminus or C-terminus to aheterologous amino acid or amino acid sequence to form a non-naturallyoccurring fusion protein. Such hIL-1Ra1S fusion variants include, forinstance, hIL-1Ra1S polypeptides wherein a heterologous secretion leadersequence is fused to the N-terminus of the sequence of amino acidresidues from at or about 47 to at or about 167 of FIG. 16 (SEQ IDNO:21), or amino acid residues from at or about 39 to at or about 167 ofFIG. 16 (SEQ ID NO:21).

Human interleukin-1 receptor antagonist analog 2 (“hIL-1Ra2”), hIL-1Ra2polypeptide, and hIL-1Ra2 protein are defined as any native sequencehIL-1Ra2 or hIL-1Ra2 fusion variant (which are further defined herein).

A “native sequence hIL-1Ra2” means (1) a polypeptide comprising theamino acid sequence of amino acid residues from at or about 1 to at orabout 134 of FIG. 5 (SEQ ID NO:10) or (2) a polypeptide consisting of anaturally-occurring truncated or secreted form of the polypeptide of(1). In one embodiment of the invention, the native sequence hIL-1Ra2consists of amino acids from at or about 27 to at or about 134 of FIG. 5(SEQ ID NO:10), or amino acids from at or about 1 to at or about 134 ofFIG. 5 (SEQ ID NO:10).

“hIL-1Ra2 fusion variant” and “hIL-1Ra2 variant” mean a chimerichIL-1Ra2 consisting of a native sequence hIL-1Ra2 fused at itsN-terminus or C-terminus to a heterologous amino acid or amino acidsequence. In one embodiment, the hIL-1Ra2 fusion variant polypeptideconsists of a native sequence of hIL-1Ra2 fused at its N-terminus orC-terminus to a heterologous amino acid or amino acid sequence, whereinthe heterologous amino acid or amino acid sequence is heterologous tothe native sequence, i.e. the resulting chimeric sequence isnon-naturally occurring. In another embodiment, the hIL-1Ra2 variantconsists of the amino acid sequence of amino acids from at or about 27to at or about 134 of FIG. 5 (SEQ ID NO:10), or amino acids from at orabout 1 to at or about 134 of FIG. 5 (SEQ ID NO:10), fused at itsN-terminus or C-terminus to a heterologous amino acid or amino acidsequence to form a non-naturally occurring fusion protein. Such hIL-1Ra2fusion variants include, for instance, hIL-1Ra2 polypeptides wherein aheterologous secretion leader sequence is fused to the N-terminus of thesequence of amino acids from at or about 27 to at or about 134 of FIG. 5(SEQ ID NO:10), or amino acids from at or about 1 to at or about 134 ofFIG. 5 (SEQ ID NO:10).

Human interleukin-1 receptor antagonist analog 3 (“hIL-1Ra3”), hIL-1Ra3polypeptide, and hIL-1Ra3 protein are defined as any native sequencehIL-1Ra3 or variant hIL-1Ra3 (which are further defined herein).

A “native sequence hIL-1Ra3” means a polypeptide comprising an aminoacid sequence selected from the group consisting of: (1) the amino acidsequence of amino acid residues from at or about 95 to at or about 134of FIG. 7 (SEQ ID NO:13); (2) the amino acid sequence of amino acidresidues from at or about 34 to at or about 155 of FIG. 7 (SEQ IDNO:13); and (3) the amino acid sequence of any naturally-occurringtruncated or secreted form or any naturally-occurring allelic variant ofa polypeptide comprising the amino acid sequence of (1) or (2). In oneembodiment of the invention, the native sequence hIL-1Ra3 comprisesamino acids from at or about 34 to at or about 155 of FIG. 7 (SEQ IDNO:13), or amino acids from at or about 2 to at or about 155 of FIG. 7(SEQ ID NO:13).

“hIL-1Ra3 variant” is defined as any hIL-1Ra3 C-terminal variant orhIL-1Ra3 full sequence variant (which are further defined herein).

“hIL-1Ra3 C-terminal variant” means any hIL-1Ra3 other than a nativesequence hIL-1Ra3, which variant is an active hIL-1Ra3, as definedbelow, having at least about 80% amino acid sequence identity with theamino acid sequence of amino acid residues from at or about 95 to at orabout 134 of FIG. 7 (SEQ ID NO:13) or the amino acid sequence of aminoacid residues from at or about 80 to at or about 155 of FIG. 7 (SEQ IDNO:13). Such hIL-1Ra3 C-terminal variants include, for instance,hIL-1Ra3 polypeptides wherein one or more amino acid residues are added,or deleted, internally or at the N- or C-terminus, in the sequence ofamino acid residues from at or about 95 to at or about 134 of FIG. 7(SEQ ID NO:13) or in the sequence of amino acid residues from at orabout 80 to at or about 155 of FIG. 7 (SEQ ID NO:13). Ordinarily, anhIL-1Ra3 C-terminal variant will have at least about 80% amino acidsequence identity, or at least about 85% amino acid sequence identity,or at least about 90% amino acid sequence identity, or at least about95% amino acid sequence identity with the amino acid sequence of aminoacid residues from at or about 95 to at or about 134 of FIG. 7 (SEQ IDNO:13) or the amino acid sequence of amino acid residues from at orabout 80 to at or about 155 of FIG. 7 (SEQ ID NO:13).

“hIL-1Ra3 full sequence variant” means any hIL-1Ra3 other than a nativesequence hIL-1Ra3, which variant retains at least one biologic activityof a native sequence hIL-1Ra3, such as the ability to bind IL-1R, andwhich variant has at least about 80% amino acid sequence identity, or atleast about 85% amino acid sequence identity, or at least about 90%amino acid sequence identity, or at least about 95% amino acid sequenceidentity with the amino acid sequence of amino acid residues from at orabout 34 to at or about 155 of FIG. 7 (SEQ ID NO:13) or the amino acidsequence of amino acid residues from at or about 2 to at or about 155 ofFIG. 7 (SEQ ID NO:13). Such hIL-1Ra3 full sequence variants include, forinstance, hIL-1Ra3 polypeptides wherein one or more amino acid residuesare added, or deleted, internally or at the N- or C-terminus, in thesequence of amino acid residues from at or about 34 to at or about 155of FIG. 7 (SEQ ID NO:13) or the amino acid sequence of amino acidresidues from at or about 2 to at or about 155 of FIG. 7 (SEQ ID NO:13).

Murine interleukin-1 receptor antagonist analog 3 (“mIL-1Ra3”), mIL-1Ra3polypeptide, and mIL-1Ra3 protein are defined as any native sequencemIL-1Ra3 or variant mIL-1Ra3.

A “native sequence mIL-1Ra3” means a polypeptide comprising an aminoacid sequence selected from the group consisting of: (1) the amino acidsequence of amino acid residues from at or about 95 to at or about 134of FIG. 9 (SEQ ID NO:16); (2) the amino acid sequence of amino acidresidues from at or about 34 to at or about 155 of FIG. 9 (SEQ IDNO:16); and (3) the amino acid sequence of any naturally-occurringtruncated or secreted form or naturally-occurring allelic variant of apolypeptide comprising the amino acid sequence of (1) or (2). In oneembodiment of the invention, the native sequence mIL-1Ra3 comprisesamino acids from at or about 34 to at or about 155 of FIG. 9 (SEQ IDNO:16).

“mIL-1Ra3 variant” is defined as any mIL-1Ra3 C-terminal variant ormIL-1Ra3 full sequence variant (which are further defined herein).

“mIL-1Ra3 C-terminal variant” means any mIL-1Ra3 other than a nativesequence mIL-1Ra3, which variant is an active mIL-1Ra3, as definedbelow, having at least about 80% amino acid sequence identity with theamino acid sequence of amino acids from at or about 95 to at or about134 of FIG. 9 (SEQ ID NO:16). Such mIL-1Ra3 C-terminal variants include,for instance, mIL-1Ra3 polypeptides wherein one or more amino acidresidues are added, or deleted, internally or at the N- or C-terminus,in the sequence of amino acids from at or about 95 to at or about 134 ofFIG. 9 (SEQ ID NO:16). Ordinarily, an mIL-1Ra3 C-terminal variant willhave at least about 80% amino acid sequence identity, or at least about85% amino acid sequence identity, or at least about 90% amino acidsequence identity, and or at least about 95% amino acid sequenceidentity with the amino acid sequence of amino acids 95 to 134 of FIG. 9(SEQ ID NO:16).

“mIL-1Ra3 full sequence variant” means any mIL-1Ra3 other than a nativesequence mIL-1Ra3, which variant retains at least one biologic activityof a native sequence mIL-1Ra3, such as the ability to bind IL-1R, andwhich variant has at least about 85% amino acid sequence identity, or atleast about 90% amino acid sequence identity, or at least about 95%sequence identity with the amino acid sequence of amino acid residuesfrom at or about 34 to at or about 155 of FIG. 9 (SEQ ID NO:16) or theamino acid sequence of amino acid residues from at or about 2 to at orabout 155 of FIG. 9 (SEQ ID NO:16). Such mIL-1Ra3 full sequence variantsinclude, for instance, mIL-1Ra3 polypeptides wherein one or more aminoacid residues are added, or deleted, internally or at the N- orC-terminus, in the sequence of amino acid residues from at or about 34to at or about 155 of FIG. 9 (SEQ ID NO:16) or in the sequence of aminoacid residues from at or about 2 to at or about 155 of FIG. 9 (SEQ IDNO:16).

“Human interleukin-1-like polypeptide”, “hIL-1lp”, “hIL-1lppolypeptide”, “hIL-1lp protein”, “human interleukin-1 receptorantagonist analog”, “hIL-1Raa”, “hIL-1Raa polypeptide”, and “hIL-1Raaprotein” are defined as any hIL-1Ra1, hIL-1Ra2 or hIL-1Ra3 polypeptide.

“Native sequence hIL-1lp” and “native sequence hIL-1Raa” are defined asany polypeptide that comprises a native sequence hIL-1Ra1, hIL-1Ra2, orhIL-1Ra3.

“hIL-1lp variant” is defined as any polypeptide that comprises a variantof hIL-1Ra1, hIL-1Ra2, or hIL-1Ra3.

“Interleukin-1 receptor”, “interleukin-1 receptor polypeptide”,“interleukin-1 receptor protein”, “IL-1 receptor”, “IL-1R”, “IL-1Rpolypeptide”, and “IL-1R protein”, are defined as the family of cellsurface proteins that bind to interleukin-1 (IL-1) and/or function inIL-1-induced signal transduction in a given species, such as human ormouse. IL-1R includes the human T cell-expressed IL-1 receptor disclosedin Sims, et al., Proc. Natl. Acad. Sci. (USA), 86: 8946-8950 (1989).

“Interleukin-18 receptor”, “interleukin-18 receptor polypeptide”,“interleukin-18 receptor protein”, “IL-18 receptor”, “IL-18R”, “IL-18Rpolypeptide”, and “IL-18R protein”, are defined as the family of cellsurface proteins that bind to interleukin-18 (IL-18) and/or function inIL-18-induced signal transduction in a given species, such as human ormouse. IL-18R includes the IL-1 receptor related protein (IL-1Rrp)described in Torigoe et al., J. Biol. Chem., 272: 25737-25742 (1997) andthe IL-18 receptor accessory protein-like molecule (IL-18RAcPL)described in Born et al., J. Biol. Chem., 273: 29445-29450 (1998).

“Interleukin-1-like family” and “IL-1-like family” are used to indicatethe family of polypeptides related to the ligands of IL-1R or IL-18R.The IL-1-like family includes IL-1 receptor agonists and antagonists andrelated polypeptides such as IL-1α (described in Bazan et al., Nature,379: 591 (1996), IL-1β (Bazan et al.), IL-18 (interferon-γ inducingfactor)(IGIF)(Bazan et al.), IL-1 receptor antagonist polypeptides suchas secretory IL-1Ra (sIL-1Ra)(described in Eisenberg et al., Nature,343: 341-346 (1990)) and intracellular IL-1Ra (icIL-1Ra) (described inHaskill et al., Proc. Natl. Acad. Sci. (USA), 88: 3681-3685 (1991)), andthe IL-1lp polypeptides of the invention.

“Percent (%) amino acid sequence identity” with respect to the IL-1lpsequences identified herein is defined as the percentage of amino acidresidues in a candidate sequence that are identical with the amino acidresidues in an IL-1lp sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign(DNASTAR) software. Those skilled in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full-length of the sequences beingcompared. For purposes herein, however, % amino acid sequence identityvalues are obtained as described below by using the sequence comparisoncomputer program ALIGN-2, wherein the complete source code for theALIGN-2 program is provided in Tables 3A-3Q. The ALIGN-2 sequencecomparison computer program was authored by Genentech, Inc. and thesource code shown in Tables 3A-3Q has been filed with user documentationin the U.S. Copyright Office, Washington D.C., 20559, where it isregistered under U.S. Copyright Registration No. TXU510087. The ALIGN-2program is publicly available through Genentech, Inc., South SanFrancisco, Calif. or may be compiled from the source code provided inTables 3A-3Q. The ALIGN-2 program should be compiled for use on a UNIXoperating system, preferably digital UNIX V4.0D. All sequence comparisonparameters are set by the ALIGN-2 program and do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which also can be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations, Tables 2A-2B demonstrate how to calculate the % amino acidsequence identity of the amino acid sequence designated “ComparisonProtein” to the amino acid sequence designated “PRO”.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described above using the ALIGN-2sequence comparison computer program. However, % amino acid sequenceidentity may also be determined using the sequence comparison programNCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).The NCBI-BLAST2 sequence comparison program may be downloaded fromhttp://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters,wherein all of those search parameters are set to default valuesincluding, for example, unmask=yes, strand=all, expected occurrences=10,minimum low complexity length=15/5, multi-pass e-value=0.01, constantfor multi-pass=25, dropoff for final gapped alignment=25 and scoringmatrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (whichalso can be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

“Percent (%) nucleic acid sequence identity” with respect to the IL-1lppolypeptide-encoding nucleic acid sequences identified herein is definedas the percentage of nucleotides in a candidate sequence that areidentical with the nucleotides in an IL-1lp polypeptide-encoding nucleicacid sequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent nucleic acid sequence identity canbe achieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % nucleic acid sequence identity values are obtained asdescribed below by using the sequence comparison computer programALIGN-2, wherein the complete source code for the ALIGN-2 program isprovided in Tables 3A-3Q. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc. and the source code shown inTables 3A-3Q has been filed with user documentation in the U.S.Copyright Office, Washington D.C., 20559, where it is registered underU.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available through Genentech, Inc., South San Francisco, Calif.or may be compiled from the source code provided in Tables 3A-3Q. TheALIGN-2 program should be compiled for use on a UNIX operating system,preferably digital UNIX V4.0D. All sequence comparison parameters areset by the ALIGN-2 program and do not vary.

For purposes herein, the % nucleic acid sequence identity of a givennucleic acid sequence C to, with, or against a given nucleic acidsequence D (which also can be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 2C-2D demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”.

Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described above using theALIGN-2 sequence comparison computer program. However, % nucleic acidsequence identity may also be determined using the sequence comparisonprogram NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402(1997)). The NCBI-BLAST2 sequence comparison program may be downloadedfrom http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several searchparameters, wherein all of those search parameters are set to defaultvalues including, for example, unmask=yes, strand=all, expectedoccurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons,the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which also can bephrased as a given nucleic acid sequence C that has or comprises acertain % nucleic acid sequence identity to, with, or against a givennucleic acid sequence D) is calculated as follows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Cand D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

The term “positives”, in the context of the amino acid sequence identitycomparisons performed as described above, includes amino acid residuesin the sequences compared that are not only identical, but also thosethat have similar properties. Amino acid residues that score a positivevalue to an amino acid residue of interest are those that are eitheridentical to the amino acid residue of interest or are a preferredsubstitution (as defined in Table 1 below) of the amino acid residue ofinterest.

For purposes herein, the % value of positives of a given amino acidsequence A to, with, or against a given amino acid sequence B (whichalso can be phrased as a given amino acid sequence A that has orcomprises a certain % positives to, with, or against a given amino acidsequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scoring a positive value asdefined above by the sequence alignment program ALIGN-2 in thatprogram's alignment of A and B, and where Y is the total number of aminoacid residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % positives of A to B will not equal the % positives of B to A.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the IL-1lp naturalenvironment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” nucleic acid molecule encoding a IL-1lp polypeptide is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the IL-1lp-encoding nucleic acid. An isolatedIL-1lp-encoding nucleic acid molecule is other than in the form orsetting in which it is found in nature. Isolated nucleic acid moleculestherefore are distinguished from the IL-1lp-encoding nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule encoding a IL-1lp polypeptide includes IL-1lp-encodingnucleic acid molecules contained in cells that ordinarily express IL-1lpwhere, for example, the nucleic acid molecule is in a chromosomallocation different from that of natural cells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers single anti-IL-1lp monoclonal antibodies (including agonist,antagonist, and neutralizing antibodies) and anti-IL-1lp antibodycompositions with polyepitopic specificity. The term “monoclonalantibody” as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising an IL-1lp polypeptide fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with activity of the polypeptide to which itis fused. The tag polypeptide preferably also is fairly unique so thatthe antibody does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least six amino acidresidues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Active” or “activity” for the purposes herein refers to form(s) ofIL-1lp which retain one or more of the biologic activities of native ornaturally-occurring IL-1lp, or which exhibit immunologicalcross-reactivity with a native or naturally-occurring IL-1lp.

As used herein, a “biologic activity” or “biological activity” of anIL-1lp means any effector function exhibited by the IL-1lp in thephysiology or pathophysiology of a mammal, excluding any immunogenic orantigenic functions of the IL-1lp. Immunogenic and antigenic functionsof an IL-1lp refer to the ability of the IL-1lp to generate a humoral orcell-mediated immune response specific to the IL-1lp, and the ability ofthe IL-1lp to specifically recognize and interact with anti-IL-1lpantibodies, B cells or T cells, respectively, in a mammal.

As used herein, “immunological cross-reactivity” with an IL-1lp meansthat the candidate polypeptide is capable of competitively inhibitingthe binding of the IL-1lp to polyclonal or monoclonal antibodies raisedagainst the IL-1lp.

In one embodiment, IL-1lp activity includes the ability to agonize orantagonize one or more biological activities of any IL-1-like familymember, e.g. an IL-1lp activity that antagonizes an IL-1-mediated orIL-18-mediated inflammatory response. In another embodiment, IL-1lpactivity includes the ability to bind to the IL-18 receptor and/or IL-1receptor.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native IL-1lp polypeptide disclosed herein. Ina similar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a nativeIL-1lp polypeptide disclosed herein. Suitable agonist or antagonistmolecules specifically include agonist or antagonist antibodies orantibody fragments, fragments or amino acid sequence variants of nativeIL-1lp polypeptides, peptides, small organic molecules, etc.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, etc.Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The terms “inflammatory disorders” and “inflammatory diseases” are usedinterchangeably herein and refer to pathological states resulting ininflammation. Examples of such disorders include inflammatory skindiseases such as psoriasis and atopic dermatitis; systemic sclerodermaand sclerosis; responses associated with inflammatory bowel disease(such as Crohn's disease and ulcerative colitis); ischemic reperfusiondisorders including surgical tissue reperfusion injury, myocardialischemic conditions such as myocardial infarction, cardiac arrest,reperfusion after cardiac surgery and constriction after percutaneoustransluminal coronary angioplasty, stroke, and abdominal aorticaneurysms; cerebral edema secondary to stroke; cranial trauma;hypovolemic shock; asphyxia; adult respiratory distress syndrome; acutelung injury; Behcet's Disease; dermatomyositis; polymyositis; multiplesclerosis; dermatitis; meningitis; encephalitis; uveitis;osteoarthritis; autoimmune diseases such as rheumatoid arthritis,Sjorgen's syndrome, vasculitis, and insulin-dependent diabetes mellitus(IDDM); diseases involving leukocyte diapedesis; central nervous system(CNS) inflammatory disorder; meningitis; multiple organ injury syndromesecondary to septicaemia or trauma; inflammatory diseases of the liver,including alcoholic hepatitis and hepatic fibrosis; pathologic hostresponses to infection, including pathologic inflammation ingranulomatous diseases, hepatitis, and bacterial pneumonia;antigen-antibody complex mediated diseases including glomerulonephritis;sepsis; sarcoidosis; immunopathologic responses to tissue/organtransplantation, including graft-versus host disease (GVHD);inflammations of the lung, including pleurisy, alveolitis, vasculitis,pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis,hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), andcystic fibrosis; inflammation in renal diseases, including acute orchronic nephritic conditions such as lupus nephritis; pancreatitis; etc.The preferred indications include rheumatoid arthritis, osteoarthritis,sepsis, acute lung injury, adult respiratory distress syndrome,idiopathic pulmonary fibrosis, ischemic reperfusion (including surgicaltissue reperfusion injury, stroke, myocardial ischemia, and acutemyocardial infarction), asthma, psoriasis, graft-versus-host disease(GVHD), and inflammatory bowel disease such as ulcerative colitis.

As used herein, the terms “asthma”, “asthmatic disorder”, “asthmaticdisease”, and “bronchial asthma” refer to a condition of the lungs inwhich there is widespread narrowing of lower airways. “Atopic asthma”and “allergic asthma” refer to asthma that is a manifestation of anIgE-mediated hypersensitivity reaction in the lower airways, including,e.g., moderate or severe chronic asthma, such as conditions requiringthe frequent or constant use of inhaled or systemic steroids to controlthe asthma symptoms. A preferred indication is allergic asthma.

II. Detailed Description of the Invention

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas IL-1lp. In particular, cDNAs encoding IL-1lp polypeptides have beenidentified and isolated, as disclosed in further detail in the Examplesbelow.

Using NCBI-BLAST2 sequence alignment computer programs, it has beenfound that a full-length native sequence hIL-1Ra1 (shown in FIG. 3 andSEQ ID NO:7) has some amino acid sequence identity with human IL-1receptor antagonist beta (hIL-1Raβ) and TANGO-77 protein, a full-lengthnative sequence hIL-1Ra1L (shown in FIG. 15 and SEQ ID NO:19) has someamino acid sequence identity with human IL-1 receptor antagonist beta(hIL-1Raβ) and TANGO-77 protein, a full-length native sequence hIL-1Ra1V(shown in FIG. 19 and SEQ ID NO:25) has some amino acid sequenceidentity with human IL-1 receptor antagonist beta (hIL-1Raβ) andTANGO-77 protein, a full-length native sequence hIL-1Ra1S (shown in FIG.16 and SEQ ID NO:21) appears to be an allelic variant of TANGO-77protein and has some amino acid sequence identity with human IL-1receptor antagonist beta (hIL-1Raβ), a full-length native sequencehIL-1Ra2 (shown in FIG. 5 and SEQ ID NO:10) has some amino acid sequenceidentity with hIL-1Raβ, a full-length native sequence hIL-1Ra3 (shown inFIG. 7 and SEQ ID NO:13) has some amino acid sequence identity withhuman intracellular IL-1 receptor antagonist (hicIL-1Ra), and afull-length native sequence mIL-1Ra3 (shown in FIG. 9 and SEQ ID NO:16)has some amino acid sequence identity with mouse IL-1 receptorantagonist (mIL-1Ra) and has some amino acid sequence identity withhicIL-1Ra. hIL-1Raβ is described in EP 0855404 published Jul. 29, 1998.TANGO-77 protein is described in WO 99/06426 published Feb. 11, 1999.hicIL-1Ra is described in WO 95/10298 published Apr. 20, 1995 and inHaskill et al., Proc. Natl. Acad. Sci. (USA), 88: 3681-3685 (1991).mIL-1Ra is described in Zahedi et al., J. Immunol., 146: 4228-4233(1991), Matsushime et al., Blood, 78: 616-623 (1991), Zahedi et al.,Cytokine, 6: 1-9 (1994), Eisenberg et al., Proc. Natl. Acad. Sci. (USA),88: 5232-5236 (1991) and Shuck et al., Eur. J. Immunol., 21: 2775-2780(1991). Accordingly, it is presently believed that the IL-1lppolypeptides disclosed in the present application are newly identifiedmembers of the interleukin-1-like family and possess inflammatory oranti-inflammatory activities, or other cellular response activating orinhibiting activities, typical of the IL-1-like family.

In addition to the full-length native sequence IL-1lp polypeptidesdescribed herein, it is contemplated that IL-1lp variants can beprepared. Such embodiments of the invention include all IL-1lppolypeptides that are IL-1lp variants as defined herein, such ashIL-1Ra1 variants, hIL-1Ra1L variants, hIL-1Ra1S variants, hIL-1Ra2variants, hIL-1Ra3 variants, and mIL-1Ra3 variants.

IL-1lp variants can be prepared by introducing appropriate nucleotidechanges into the IL-1lp DNA, and/or by synthesis of the desired IL-1lppolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the IL-1lp, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

Variations in the native full-length sequence IL-1lp or in variousdomains of the IL-1lp described herein, can be made, for example, usingany of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding the IL-1lp that results in a change in theamino acid sequence of the IL-1lp as compared with the native sequenceIL-1lp. Optionally the variation is by substitution of at least oneamino acid with any other amino acid in one or more of the domains ofthe IL-1lp. Guidance in determining which amino acid residue may beinserted, substituted or deleted without adversely affecting the desiredactivity may be found by comparing the sequence of the IL-1lp with thatof homologous known protein molecules and minimizing the number of aminoacid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions may optionally be in therange of 1 to 5 amino acids. The variation allowed may be determined bysystematically making insertions, deletions or substitutions of aminoacids in the sequence and testing the resulting variants for activity inthe in vitro assay described in the Examples below.

Table 1 below lists conservative amino acid substitutions (under theheading of “Preferred Substitutions”) that are useful in generatingvariants of the native sequence IL-1lp. If such substitutions result inalteration of biological activity, it is useful to introduce moresubstantial changes, such as the “Exemplary Substitutions” denoted inTable 1 or the substantial changes described below in reference to aminoacid classes, at the active site in question. TABLE 1 Original ExemplaryPreferred Residue Substitutions Substitutions Ala (A) val; leu; ile valArg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu gluCys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His(H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leunorleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys (K) arg;gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyrleu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyrTyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala;norleucine

Substantial modifications in function or immunological identity of theIL-1lp polypeptide are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)),restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the IL-1lp variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

Covalent modifications of IL-1lp are included within the scope of thisinvention. One type of covalent modification includes reacting targetedamino acid residues of an IL-1lp polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues of the IL-1lp. Derivatization withbifunctional agents is useful, for instance, for crosslinking IL-1lp toa water-insoluble support matrix or surface for use in the method forpurifying anti-IL-1lp antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the IL-1lp polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence IL-1lp(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequenceIL-1lp. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

Addition of glycosylation sites to the IL-1lp polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence IL-1lp (for O-linkedglycosylation sites). The IL-1lp amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the IL-1lp polypeptide at preselected bases such thatcodons are generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theIL-1lp polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the IL-1lp polypeptide maybe accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification of IL-1lp comprises linking theIL-1lp polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The IL-1lp of the present invention may also be modified in a way toform a chimeric molecule comprising IL-1lp fused to another,heterologous polypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of theIL-1lp with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the IL-1lp. The presence ofsuch epitope-tagged forms of the IL-1lp can be detected using anantibody against the tag polypeptide. Also, provision of the epitope tagenables the IL-1lp to be readily purified by affinity purification usingan anti-tag antibody or another type of affinity matrix that binds tothe epitope tag. Various tag polypeptides and their respectiveantibodies are well known in the art. Examples include poly-histidine(poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tagpolypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.,8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the IL-1lp with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an immunoadhesin), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble form of an IL-1lp polypeptide in place of atleast one variable region within an Ig molecule. In a particularlypreferred embodiment, the immunoglobulin fusion includes the hinge, CH2and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. Forthe production of immunoglobulin fusions see also U.S. Pat. No.5,428,130 issued Jun. 27, 1995.

In one aspect, the invention provides an isolated nucleic acidcomprising DNA encoding an IL-1lp polypeptide that retains at least onebiologic activity of a native sequence IL-1lp, such as the IL-1R bindingactivity of a native sequence hIL-1Ra3 or mIL-1Ra3, or the IL-18Rbinding activity of a native sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V,which DNA has at least at or about 80% sequence identity, or at least ator about 85% sequence identity, or at least at or about 90% sequenceidentity, or at least at or about 95% sequence identity to (a) a DNAmolecule selected from the group consisting of: (1) a DNA moleculeencoding an IL-1lp polypeptide comprising amino acid residues from at orabout 37 to at or about 203, inclusive of FIG. 2 (SEQ ID NO:5), (2) aDNA molecule encoding an IL-1lp polypeptide comprising amino acidresidues from at or about 15 to at or about 193, inclusive of FIG. 3(SEQ ID NO:7), (3) a DNA molecule encoding an IL-1lp polypeptidecomprising amino acid residues from at or about 34 to at or about 155,inclusive of FIG. 7 (SEQ ID NO:13), (4) a DNA molecule encoding anIL-1lp polypeptide comprising amino acid residues from at or about 34 toat or about 155, inclusive of FIG. 9 (SEQ ID NO:16), (5) a DNA moleculeencoding an IL-1lp polypeptide comprising amino acid residues from at orabout 26 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19), and(6) a DNA molecule encoding an IL-1lp polypeptide comprising amino acidresidues from at or about 46 to at or about 218 of FIG. 19 (SEQ IDNO:25), or (b) the complement of the DNA molecule of (a).

In another aspect, the invention provides an isolated nucleic acidcomprising DNA encoding an IL-1lp polypeptide that retains at least onebiologic activity of a native sequence IL-1lp, such as the IL-18Rbinding activity of a native sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V,which DNA has at least at or about 80% sequence identity, or at least ator about 85% sequence identity, or at least at or about 90% sequenceidentity, or at least at or about 95% sequence identity to (a) a DNAmolecule selected from the group consisting of: (1) a DNA moleculeencoding an IL-1lp polypeptide comprising amino acid residues from at orabout 1 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19), and (2)a DNA molecule encoding an IL-1lp polypeptide comprising amino acidresidues from at or about 1 to at or about 218 of FIG. 19 (SEQ IDNO:25), or (b) the complement of the DNA molecule of (a).

In another aspect, the invention provides an isolated nucleic acidcomprising DNA encoding an IL-1lp polypeptide that retains at least onebiologic activity of a native sequence IL-1lp, such as the IL-18Rbinding activity of a native sequence hIL-1Ra1, or the IL-1R bindingactivity of a native sequence hIL-1Ra3 or mIL-1Ra3, which DNA has atleast at or about 80% sequence identity, or at least at or about 85%sequence identity, or at least at or about 90% sequence identity, or atleast at or about 95% sequence identity to (a) a DNA molecule selectedfrom the group consisting of: (1) a DNA molecule encoding an IL-1lppolypeptide comprising amino acid residues from at or about 95 to at orabout 134, inclusive of FIG. 7 (SEQ ID NO:13), and (2) a DNA moleculeencoding an IL-1lp polypeptide comprising amino acid residues from at orabout 95 to at or about 134, inclusive of FIG. 9 (SEQ ID NO:16), or (b)the complement of the DNA molecule of (a).

In another aspect, the invention provides an isolated nucleic acidcomprising DNA encoding an IL-1lp polypeptide that retains at least onebiologic activity of a native sequence IL-1lp, such as the IL-18Rbinding activity of a native sequence hIL-1Ra1, or the IL-1R bindingactivity of a native sequence hIL-1Ra3 or mIL-1Ra3, which DNA has atleast at or about 80% sequence identity, or at least at or about 85%sequence identity, or at least at or about 90% sequence identity, or atleast at or about 95% sequence identity to (a) a DNA molecule encodingan IL-1lp polypeptide comprising amino acid residues from at or about 80to at or about 155, inclusive of FIG. 7 (SEQ ID NO:13), or (b) thecomplement of the DNA molecule of (a).

In another aspect, the invention provides an isolated nucleic acidcomprising DNA encoding an IL-1lp polypeptide that retains at least onebiologic activity of a native sequence IL-1lp, such as the IL-18Rbinding activity of a native sequence hIL-1Ra1, or the IL-1R bindingactivity of a native sequence hIL-1Ra3 or mIL-1Ra3, which DNA has atleast at or about 80% sequence identity, or at least at or about 85%sequence identity, or at least at or about 90% sequence identity, or atleast at or about 95% sequence identity to (a) a DNA molecule selectedfrom the group consisting of: (1) a DNA molecule encoding an IL-1lppolypeptide comprising amino acid residues from at or about 2 to at orabout 155, inclusive of FIG. 7 (SEQ ID NO:13), and (2) a DNA moleculeencoding an IL-1lp polypeptide comprising amino acid residues from at orabout 2 to at or about 155, inclusive of FIG. 9 (SEQ ID NO:16), or (b)the complement of the DNA molecule of (a).

In another aspect, the invention provides an isolated nucleic acidcomprising DNA having at least at or about 80% sequence identity, or atleast at or about 85% sequence identity, or at least at or about 90%sequence identity, or at least at or about 95% sequence identity to (a)a DNA molecule selected from the group consisting of: (1) a DNA moleculeencoding an IL-1lp polypeptide comprising amino acid residues from at orabout 95 to at or about 134, inclusive of FIG. 7 (SEQ ID NO:13), and (2)a DNA molecule encoding an IL-1lp polypeptide comprising amino acidresidues from at or about 95 to at or about 134, inclusive of FIG. 9(SEQ ID NO:16), or (b) the complement of the DNA molecule of (a).

In another aspect, the invention provides an isolated nucleic acidcomprising DNA having at least at or about 80% sequence identity, or atleast at or about 85% sequence identity, or at least at or about 90%sequence identity, or at least at or about 95% sequence identity to (a)a DNA molecule encoding an IL-1lp polypeptide comprising amino acidresidues from at or about 80 to at or about 155, inclusive of FIG. 7(SEQ ID NO:13), or (b) the complement of the DNA molecule of (a).

In another aspect, the invention provides an isolated nucleic acidcomprising DNA having at least at or about 80% sequence identity, or atleast at or about 85% sequence identity, or at least at or about 90%sequence identity, or at least at or about 95% sequence identity to (a)a DNA molecule selected from the group consisting of: (1) a DNA moleculeencoding an IL-1lp polypeptide comprising amino acid residues from at orabout 2 to at or about 155, inclusive of FIG. 7 (SEQ ID NO:13), and (2)a DNA molecule encoding an IL-1lp polypeptide comprising amino acidresidues from at or about 2 to at or about 155, inclusive of FIG. 9 (SEQID NO:16), or (b) the complement of the DNA molecule of (a).

In another aspect, the invention concerns an isolated nucleic acidmolecule encoding an IL-1lp polypeptide, comprising DNA hybridizing tothe complement of a nucleic acid sequence selected from the groupconsisting of: (1) the nucleic acid sequence consisting of nucleotidepositions from at or about 238 to at or about 465 in the sense strand ofFIG. 7 (SEQ ID NO:12); (2) the nucleic acid sequence consisting ofnucleotide positions from at or about 427 to at or about 609 in thesense strand of FIG. 9 (SEQ ID NO:15); and (3) the nucleic acid sequenceconsisting of nucleotide positions from at or about 79 to at or about135 in the sense strand of FIG. 15 (SEQ ID NO:18). Preferably,hybridization occurs under stringent hybridization and wash conditions.

In another aspect, the invention concerns an isolated nucleic acidmolecule, comprising DNA that is at least 90 nucleotides in length andthat hybridizes to the complement of a nucleic acid sequence selectedfrom the group consisting of: (1) the nucleic acid sequence consistingof nucleotide positions from at or about 238 to at or about 465 in thesense strand of FIG. 7 (SEQ ID NO:12); (2) the nucleic acid sequenceconsisting of nucleotide positions from at or about 427 to at or about609 in the sense strand of FIG. 9 (SEQ ID NO:15); and (3) the nucleicacid sequence consisting of nucleotide positions from at or about 115 toat or about 135 in the sense strand of FIG. 15 (SEQ ID NO:18).Preferably, hybridization occurs under stringent hybridization and washconditions.

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA encoding an IL-1lp polypeptide that retains atleast one biologic activity of a native sequence IL-1lp, such as theIL-1R binding activity of a native sequence hIL-1Ra3 or mIL-1Ra3, or theIL-18R binding activity of a native sequence hIL-1Ra1, hIL-1Ra1L, orhIL-1Ra1V, which DNA hybridizes to the complement of a nucleic acidsequence selected from the group consisting of: (1) the nucleic acidsequence consisting of nucleotide positions from at or about 118 to ator about 231 in the sense strand of FIG. 2 (SEQ ID NO:4); (2) thenucleic acid sequence consisting of nucleotide positions from at orabout 100 to at or about 465 in the sense strand of FIG. 7 (SEQ IDNO:12); (3) the nucleic acid sequence consisting of nucleotide positionsfrom at or about 244 to at or about 609 in the sense strand of FIG. 9(SEQ ID NO:15); and (4) the nucleic acid sequence consisting ofnucleotide positions from at or about 208 to at or about 339 in thesense strand of FIG. 19 (SEQ ID NO:24). Preferably, hybridization occursunder stringent hybridization and wash conditions.

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA encoding an IL-1lp polypeptide that retains atleast one biologic activity of a native sequence IL-1lp, such as theIL-1R binding activity of a native sequence hIL-1Ra3 or mIL-1Ra3, whichDNA hybridizes to the complement of a nucleic acid sequence selectedfrom the group consisting of: (1) the nucleic acid sequence consistingof nucleotide positions from at or about 4 to at or about 465 in thesense strand of FIG. 7 (SEQ ID NO:12); and (2) the nucleic acid sequenceconsisting of nucleotide positions from at or about 148 to at or about609 in the sense strand of FIG. 9 (SEQ ID NO:15). Preferably,hybridization occurs under stringent hybridization and wash conditions.

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA encoding an IL-1lp polypeptide that retains atleast one biologic activity of a native sequence IL-1lp, such as theIL-1R binding activity of a native sequence hIL-1Ra3 or mIL-1Ra3, or theIL-18R binding activity of a native sequence hIL-1Ra1, hIL-1Ra1L, orhIL-1Ra1V, which DNA has at least at or about 80% sequence identity, orat least at or about 85% sequence identity, or at least at or about 90%sequence identity, or at least at or about 95% sequence identity to (a)a DNA encoding an IL-1lp, such as a mature IL-1lp polypeptide, encodedby the cDNA insert in the vector deposited as ATCC Deposit No. 203588(DNA85066-2534), ATCC Deposit No. 203587 (DNA96786-2534), ATCC DepositNo. 203589 (DNA96787-2534), ATCC Deposit No. 203590 (DNA92505-2534),ATCC Deposit No. 203846 (DNA102043-2534), or ATCC Deposit No. 203973(DNA114876-2534), or (b) the complement of the DNA molecule of (a). In apreferred embodiment, the nucleic acid comprises a DNA encoding anIL-1lp polypeptide, such as a mature IL-1lp polypeptide, encoded by thecDNA insert in the vector deposited as ATCC Deposit No. 203588(DNA85066-2534), ATCC Deposit No. 203587 (DNA96786-2534), ATCC DepositNo. 203586 (DNA92929-2534), ATCC Deposit No. 203589 (DNA96787-2534),ATCC Deposit No. 203590 (DNA92505-2534), ATCC Deposit No. 203846(DNA102043-2534), ATCC Deposit No. 203973 (DNA1 14876-2534), or ATCCDeposit No. 203855 (DNA102044-2534).

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA encoding an IL-1lp polypeptide that retains atleast one biologic activity of a native sequence IL-1lp, such as theIL-1R binding activity of a native sequence hIL-1Ra3 or mIL-1Ra3, or theIL-18R binding activity of a native sequence hIL-1Ra1, hIL-1Ra1L, orhIL-1Ra1V, which DNA has at least at or about 80% sequence identity, orat least at or about 85% sequence identity, or at least at or about 90%sequence identity, or at least at or about 95% sequence identity to (a)a DNA encoding the entire amino acid sequence encoded by the longestopen reading frame in the cDNA insert of a vector selected from thegroup consisting of the vectors deposited as ATCC Deposit No. 203588(DNA85066-2534), ATCC Deposit No. 203587 (DNA96786-2534), ATCC DepositNo. 203589 (DNA96787-2534), ATCC Deposit No. 203590 (DNA92505-2534),ATCC Deposit No. 203846 (DNA102043-2534), and ATCC Deposit No. 203973(DNA114876-2534), or (b) the complement of the DNA molecule of (a). In apreferred embodiment, the nucleic acid comprises (a) DNA encoding theentire amino acid sequence encoded by the longest open reading frame inthe cDNA insert of a vector selected from the group consisting of thevectors deposited as ATCC Deposit No. 203588 (DNA85066-2534), ATCCDeposit No. 203587 (DNA96786-2534), ATCC Deposit No. 203586(DNA92929-2534), ATCC Deposit No. 203589 (DNA96787-2534), and ATCCDeposit No. 203590 (DNA92505-2534), or (b) the complement of the DNA of(a). In another preferred embodiment, the nucleic acid comprises (a) DNAencoding the entire amino acid sequence encoded by the longest openreading frame in the cDNA insert of a vector selected from the groupconsisting of the vectors deposited as ATCC Deposit No. 203846(DNA102043-2534), ATCC Deposit No. 203855 (DNA102044-2534), and ATCCDeposit No. 203973 (DNA114876-2534), or (b) the complement of the DNA of(a).

In another aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA encoding an IL-1lp polypeptide that retains atleast one biologic activity of a native sequence IL-1lp, such as theIL-1R binding activity of a native sequence IL-1Ra3 or mIL-1Ra3, or theIL-18R binding activity of a native sequence hIL-1Ra1, hIL-1Ra1L, orhIL-1Ra1V, which DNA has at least at or about 80% sequence identity, orat least at or about 85% sequence identity, or at least at or about 90%sequence identity, or at least at or about 95% sequence identity to (a)DNA encoding an amino acid sequence selected from the group consistingof: (1) the entire amino acid sequence encoded by the longest openreading frame in the cDNA insert in the vector deposited as ATCC DepositNo. 203588, (2) the entire amino acid sequence, or the entire amino acidsequence excluding the 36 N-terminal amino acid residues of suchsequence, encoded by the longest open reading frame in the cDNA insertin the vector deposited as ATCC Deposit No. 203587, (3) the entire aminoacid sequence, or the entire amino acid sequence excluding theN-terminal amino acid residue of such sequence, encoded by the longestopen reading frame in the cDNA insert in the vector deposited as ATCCDeposit No. 203589, (4) the entire amino acid sequence, or the entireamino acid sequence excluding the N-terminal amino acid residue of suchsequence, encoded by the longest open reading frame in the cDNA insertin the vector deposited as ATCC Deposit No. 203590, (5) the entire aminoacid sequence, or the entire amino acid sequence excluding theN-terminal amino acid residue of such sequence, or the entire amino acidsequence excluding the 34 N-terminal amino acid residues of suchsequence, encoded by the longest open reading frame in the cDNA insertin the vector deposited as ATCC Deposit No. 203846, and (6) the entireamino acid sequence, or the entire amino acid sequence excluding theN-terminal amino acid residue of such sequence, or the entire amino acidsequence excluding the 45 N-terminal amino acid residues of suchsequence, encoded by the longest open reading frame in the cDNA insertin the vector deposited as ATCC Deposit No. 203973, or (b) thecomplement of the DNA of (a).

In a preferred embodiment, the nucleic acid comprises (a) DNA encodingan amino acid sequence selected from the group consisting of: (1) theentire amino acid sequence encoded by the longest open reading frame inthe cDNA insert in the vector deposited as ATCC Deposit No. 203588, (2)the entire amino acid sequence, or the entire amino acid sequenceexcluding the 36 N-terminal amino acid residues of such sequence,encoded by the longest open reading frame in the cDNA insert in thevector deposited as ATCC Deposit No. 203587, (3) the entire amino acidsequence, or the entire amino acid sequence excluding the N-terminalamino acid residue of such sequence, encoded by the longest open readingframe in the cDNA insert in the vector deposited as ATCC Deposit No.203589, (4) the entire amino acid sequence, or the entire amino acidsequence excluding the N-terminal amino acid residue of such sequence,encoded by the longest open reading frame in the cDNA insert in thevector deposited as ATCC Deposit No. 203590, (5) the entire amino acidsequence, or the entire amino acid sequence excluding the N-terminalamino acid residue of such sequence, or the entire amino acid sequenceexcluding the 34 N-terminal amino acid residues of such sequence,encoded by the longest open reading frame in the cDNA insert in thevector deposited as ATCC Deposit No. 203846, and (6) the entire aminoacid sequence, or the entire amino acid sequence excluding theN-terminal amino acid residue of such sequence, or the entire amino acidsequence excluding the 45 N-terminal amino acid residues of suchsequence, encoded by the longest open reading frame in the cDNA insertin the vector deposited as ATCC Deposit No. 203973, or (b) thecomplement of the DNA of (a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide thatretains at least one biologic activity of a native sequence IL-1lp, suchas the IL-18R binding activity of a native sequence hIL-1Ra1, or theIL-1R binding activity of a native sequence hIL-1Ra3 or mIL-1Ra3, andwhich IL-1lp polypeptide has at least at or about 80% sequence identity,or at least at or about 85% sequence identity, or at least at or about90% sequence identity, or at least at or about 95% sequence identity toan amino acid sequence selected from the group consisting of: (1) aminoacid residues from at or about 37 to at or about 203, inclusive of FIG.2 (SEQ ID NO:5), (2) amino acid residues from at or about 15 to at orabout 193, inclusive of FIG. 3 (SEQ ID NO:7), (3) amino acid residuesfrom at or about 34 to at or about 155, inclusive of FIG. 7 (SEQ IDNO:13), (4) amino acid residues from at or about 34 to at or about 155,inclusive of FIG. 9 (SEQ ID NO:16), (5) amino acid residues from at orabout 26 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19), and(6) amino acid residues from at or about 46 to at or about 218,inclusive of FIG. 19 (SEQ ID NO:25), or (b) the complement of the DNA of(a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide thatretains at least one biologic activity of a native sequence IL-1lp, suchas the IL-18R binding activity of a native sequence hIL-1Ra1, or theIL-1R binding activity of a native sequence hIL-1Ra3 or mIL-1Ra3, andwhich IL-1lp polypeptide has at least at or about 80% sequence identity,or at least at or about 85% sequence identity, or at least at or about90% sequence identity, or at least at or about 95% sequence identity toan amino acid sequence selected from the group consisting of: (1) aminoacid residues from at or about 95 to at or about 134, inclusive of FIG.7 (SEQ ID NO:13), and (2) amino acid residues from at or about 95 to ator about 134, inclusive of FIG. 9 (SEQ ID NO:16), or (b) the complementof the DNA of (a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide thatretains at least one biologic activity of a native sequence IL-1lp, suchas the IL-18R binding activity of a native sequence hIL-1Ra1, or theIL-1R binding activity of a native sequence hIL-1Ra3 or mIL-1Ra3, andwhich IL-1lp polypeptide has at least at or about 80% sequence identity,or at least at or about 85% sequence identity, or at least at or about90% sequence identity, or at least at or about 95% sequence identity tothe amino acid sequence of amino acid residues from at or about 80 to ator about 155, inclusive of FIG. 7 (SEQ ID NO:13), or (b) the complementof the DNA of (a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide thatretains at least one biologic activity of a native sequence IL-1lp, suchas the IL-18R binding activity of a native sequence hIL-1Ra1, or theIL-1R binding activity of a native sequence hIL-1Ra3 or mIL-1Ra3, andwhich IL-1lp polypeptide has at least at or about 80% sequence identity,or at least at or about 85% sequence identity, or at least at or about90% sequence identity, or at least at or about 95% sequence identity toan amino acid sequence selected from the group consisting of: (1) aminoacid residues from at or about 2 to at or about 155, inclusive of FIG. 7(SEQ ID NO:13), and (2) amino acid residues from at or about 2 to at orabout 155, inclusive of FIG. 9 (SEQ ID NO:16), or (b) the complement ofthe DNA of (a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide havingat least at or about 80% sequence identity, or at least at or about 85%sequence identity, or at least at or about 90% sequence identity, or atleast at or about 95% sequence identity to an amino acid sequenceselected from the group consisting of: (1) amino acid residues from ator about 95 to at or about 134, inclusive of FIG. 7 (SEQ ID NO:13), and(2) amino acid residues from at or about 95 to at or about 134,inclusive of FIG. 9 (SEQ ID NO:16), or (b) the complement of the DNA of(a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide havingat least at or about 80% sequence identity, or at least at or about 85%sequence identity, or at least at or about 90% sequence identity, or atleast at or about 95% sequence identity to the amino acid sequence ofamino acid residues from at or about 80 to at or about 155, inclusive ofFIG. 7 (SEQ ID NO:13), or (b) the complement of the DNA of (a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide havingat least at or about 80% sequence identity, or at least at or about 85%sequence identity, or at least at or about 90% sequence identity, or atleast at or about 95% sequence identity to an amino acid sequenceselected from the group consisting of: (1) amino acid residues from ator about 2 to at or about 155, inclusive of FIG. 7 (SEQ ID NO:13), and(2) amino acid residues from at or about 2 to at or about 155, inclusiveof FIG. 9 (SEQ ID NO:16), or (b) the complement of the DNA of (a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide thatretains at least one biologic activity of a native sequence IL-1lp, suchas the IL-18R binding activity of a native sequence hIL-1Ra1, hIL-1Ra1L,or hIL-1Ra1V, and which IL-1lp polypeptide has at least at or about 80%sequence identity, or at least at or about 85% sequence identity, or atleast at or about 90% sequence identity, or at least at or about 95%sequence identity to an amino acid sequence selected from the groupconsisting of: (1) amino acid residues from at or about 1 to at or about207, inclusive of FIG. 15 (SEQ ID NO:19), and (2) amino acid residuesfrom at or about 1 to at or about 218, inclusive of FIG. 19 (SEQ IDNO:25), or (b) the complement of the DNA of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising DNA encoding an IL-1lp that retains at least onebiologic activity of a native sequence IL-1lp, such as the IL-1R bindingactivity of a native sequence hIL-1Ra3 or mIL-1Ra3, or the IL-18Rbinding activity of a native sequence hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1S, orhIL-1Ra1V, which DNA is produced by hybridizing a test DNA moleculeunder stringent conditions with (a) a DNA molecule encoding an IL-1lppolypeptide selected from the group consisting of: (1) an IL-1lppolypeptide comprising the sequence of amino acid residues from at orabout 37 to at or about 203, inclusive of FIG. 2 (SEQ ID NO:5), (2) anIL-1lp polypeptide comprising the sequence of amino acid residues fromat or about 15 to at or about 193, inclusive of FIG. 3 (SEQ ID NO:7),(3) an IL-1lp polypeptide comprising the sequence of amino acid residuesfrom at or about 34 to at or about 155, inclusive of FIG. 7 (SEQ IDNO:13), (4) an IL-1lp polypeptide comprising the sequence of amino acidresidues from at or about 34 to at or about 155, inclusive of FIG. 9(SEQ ID NO:16), (5) an IL-1lp polypeptide comprising the sequence ofamino acid residues from at or about 26 to at or about 207, inclusive ofFIG. 15 (SEQ ID NO:19), and (6) an IL-1lp polypeptide comprising thesequence of amino acid residues from at or about 46 to at or about 218,inclusive of FIG. 19 (SEQ ID NO:25), or (b) the complement of the DNAmolecule of (a), and, if the test DNA molecule encodes an IL-1lp thatretains at least one biologic activity of a native sequence IL-1lp, suchas the IL-1R binding activity of a native sequence hIL-1Ra3 or mIL-1Ra3,or the IL-18R binding activity of a native sequence hIL-1Ra1, hIL-1Ra1L,or hIL-1Ra1V, and if the test DNA molecule has at least at or about an80% sequence identity, or at least at or about an 85% sequence identity,or at least at or about a 90% sequence identity, or at least at or abouta 95% sequence identity to the DNA molecule of (a) or (b), isolating thetest DNA molecule.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising (a) DNA encoding an a polypeptide, such as IL-1lppolypeptide, selected from the group consisting of: (1) a polypeptide,such as an hIL-1Ra1 polypeptide, comprising amino acid residues from ator about 37 to at or about 63, inclusive of FIG. 2 (SEQ ID NO:5); (2) apolypeptide, such as an hIL-1Ra1 polypeptide, comprising amino acidresidues from at or about 15 to at or about 53, inclusive of FIG. 3 (SEQID NO:7); (3) a polypeptide, such as an hIL-1Ra2 polypeptide, comprisingamino acid residues from at or about 1 to at or about 134, inclusive ofFIG. 5 (SEQ ID NO:10); (4) a polypeptide comprising amino acid residuesfrom at or about 10 to at or about 134, inclusive of FIG. 5 (SEQ IDNO:10); (5) a polypeptide, such as an hIL-1Ra2 polypeptide, consistingof amino acid residues from at or about 27 to at or about 134, inclusiveof FIG. 5 (SEQ ID NO:10); (6) a polypeptide, such as an hIL-1Ra2 fusionvariant polypeptide, consisting of a native amino acid sequence ofhIL-1Ra2 consisting of amino acid residues from at or about 27 to at orabout 134, inclusive of FIG. 5 (SEQ ID NO:10) fused at its N-terminus orC-terminus to a heterologous amino acid or amino acid sequence; (7) apolypeptide, such as an hIL-1Ra3 polypeptide, comprising amino acidresidues from at or about 95 to at or about 134, inclusive of FIG. 7(SEQ ID NO:13); and (8) a polypeptide, such as a mIL-1Ra3 polypeptide,comprising amino acid residues from at or about 95 to at or about 134,inclusive of FIG. 9 (SEQ ID NO:16); or (b) the complement of the DNA of(a).

In another aspect, the invention provides an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide, such as an IL-1lppolypeptide, selected from the group consisting of: (1) a polypeptide,such as an hIL-1Ra1L polypeptide, comprising amino acid residues from ator about 26 to at or about 44, inclusive of FIG. 15 (SEQ ID NO:19); (2)a polypeptide, such as an hIL-1Ra1L polypeptide, comprising amino acidresidues from at or about 1 to at or about 44, inclusive of FIG. 15 (SEQID NO:19); (3) a polypeptide, such as an hIL-1Ra1L polypeptide,comprising amino acid residues from at or about 26 to at or about 78,inclusive of FIG. 15 (SEQ ID NO:19); (4) a polypeptide, such as anhIL-1Ra1L polypeptide, comprising amino acid residues from at or about 1to at or about 78, inclusive of FIG. 15 (SEQ ID NO:19); (5) apolypeptide, such as an hIL-1Ra1S polypeptide, comprising amino acidresidues from at or about 1 to at or about 38, inclusive of FIG. 16 (SEQID NO:21); (6) a polypeptide, such as an hIL-1Ra1V polypeptide,comprising amino acid residues from at or about 37 to at or about 55,inclusive of FIG. 19 (SEQ ID NO:25); (7) a polypeptide, such as anhIL-1Ra1V polypeptide, comprising amino acid residues from at or about12 to at or about 55, inclusive of FIG. 19 (SEQ ID NO:25); (8) apolypeptide, such as an hIL-1Ra1V polypeptide, comprising amino acidresidues from at or about 1 to at or about 55, inclusive of FIG. 19 (SEQID NO:25); (9) a polypeptide, such as an hIL-1Ra1V polypeptide,comprising amino acid residues from at or about 46 to at or about 55,inclusive of FIG. 19 (SEQ ID NO:25); (10) a polypeptide, such as anhIL-1Ra1V polypeptide, comprising amino acid residues from at or about46 to at or about 89, inclusive of FIG. 19 (SEQ ID NO:25); (11) apolypeptide, such as an hIL-1Ra1V polypeptide, comprising amino acidresidues from at or about 37 to at or about 89, inclusive of FIG. 19(SEQ ID NO:25); (12) a polypeptide, such as an hIL-1Ra1V polypeptide,comprising amino acid residues from at or about 12 to at or about 89,inclusive of FIG. 19 (SEQ ID NO:25); and (13) a polypeptide, such as anhIL-1Ra1V polypeptide, comprising amino acid residues from at or about 1to at or about 89, inclusive of FIG. 19 (SEQ ID NO:25); or (b) thecomplement of the DNA of (a).

In another aspect, the invention provides an isolated nucleic acidmolecule comprising (a) DNA encoding a polypeptide, such as an hIL-1lppolypeptide, selected from the group consisting of: (1) a polypeptide,such as an hIL-1Ra1L polypeptide, consisting of a native amino acidsequence of hIL-1Ra1L consisting of amino acid residues from at or about26 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19) fused at itsN-terminus or C-terminus to a heterologous amino acid or amino acidsequence; (2) a polypeptide, such as an hIL-1Ra1L polypeptide,consisting of a native amino acid sequence of hIL-1Ra1L consisting ofamino acid residues from at or about 1 to at or about 207, inclusive ofFIG. 15 (SEQ ID NO:19) fused at its N-terminus or C-terminus to aheterologous amino acid or amino acid sequence; (3) a polypeptide, suchas an hIL-1Ra1L polypeptide, consisting of amino acid residues from ator about 26 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19); (4)a polypeptide, such as an hIL-1Ra1L polypeptide, consisting of aminoacid residues from at or about 1 to at or about 207, inclusive of FIG.15 (SEQ ID NO:19); (5) a polypeptide, such as an hIL-1Ra1S fusionvariant polypeptide, consisting of a native amino acid sequence ofhIL-1Ra1S consisting of amino acid residues from at or about 26 to at orabout 167, inclusive of FIG. 16 (SEQ ID NO:21) fused at its N-terminusor C-terminus to a heterologous amino acid or amino acid sequence; (6) apolypeptide, such as an hIL-1Ra1S polypeptide, consisting of amino acidresidues from at or about 26 to at or about 167, inclusive of FIG. 16(SEQ ID NO:21); (7) a polypeptide, such as an hIL-1Ra1S fusion variantpolypeptide, consisting of a native amino acid sequence of hIL-1Ra1Sconsisting of amino acid residues from at or about 1 to at or about 167,inclusive of FIG. 16 (SEQ ID NO:21) fused at its N-terminus orC-terminus to a heterologous amino acid or amino acid sequence; (8) apolypeptide, such as an hIL-1Ra1S polypeptide, consisting of amino acidresidues from at or about 1 to at or about 167, inclusive of FIG. 16(SEQ ID NO:21); (9) a polypeptide, such as an hIL-1Ra1S fusion variantpolypeptide, consisting of a native amino acid sequence of hIL-1Ra1Sconsisting of amino acid residues from at or about 39 to at or about167, inclusive of FIG. 16 (SEQ ID NO:21) fused at its N-terminus orC-terminus to a heterologous amino acid or amino acid sequence; (10) apolypeptide, such as an hIL-1Ra1S fusion variant polypeptide, consistingof a native amino acid sequence of hIL-1Ra1S consisting of amino acidresidues from at or about 47 to at or about 167, inclusive of FIG. 16(SEQ ID NO:21) fused at its N-terminus or C-terminus to a heterologousamino acid or amino acid sequence; (11) a polypeptide, such as anhIL-1Ra1S polypeptide, consisting of amino acid residues from at orabout 39 to at or about 167, inclusive of FIG. 16 (SEQ ID NO:21); (12) apolypeptide, such as an hIL-1Ra1S polypeptide, consisting of amino acidresidues from at or about 47 to at or about 167, inclusive of FIG. 16(SEQ ID NO:21); (13) a polypeptide, such as an hIL-1Ra1V polypeptide,consisting of a native amino acid sequence of hIL-1Ra1V consisting ofamino acid residues from at or about 1 to at or about 218, inclusive ofFIG. 19 (SEQ ID NO:25) fused at its N-terminus or C-terminus to aheterologous amino acid or amino acid sequence; (14) a polypeptide, suchas an hIL-1Ra1V polypeptide, consisting of amino acid residues from ator about 1 to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25); (15)a polypeptide, such as an hIL-1Ra1V polypeptide, consisting of a nativeamino acid sequence of hIL-1Ra1V consisting of amino acid residues fromat or about 12 to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25)fused at its N-terminus or C-terminus to a heterologous amino acid oramino acid sequence; (16) a polypeptide, such as an hIL-1Ra1Vpolypeptide, consisting of amino acid residues from at or about 12 to ator about 218, inclusive of FIG. 19 (SEQ ID NO:25); (17) a polypeptide,such as an hIL-1Ra1V fusion variant polypeptide, consisting of a nativeamino acid sequence of hIL-1Ra1V consisting of amino acid residues fromat or about 37 to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25)fused at its N-terminus or C-terminus to a heterologous amino acid oramino acid sequence; (18) a polypeptide, such as an hIL-1Ra1V fusionvariant polypeptide, consisting of a native amino acid sequence ofhIL-1Ra1V consisting of amino acid residues from at or about 46 to at orabout 218, inclusive of FIG. 19 (SEQ ID NO:25) fused at its N-terminusor C-terminus to a heterologous amino acid or amino acid sequence; (19)a polypeptide, such as an hIL-1Ra1V polypeptide, consisting of aminoacid residues from at or about 37 to at or about 218, inclusive of FIG.19 (SEQ ID NO:25); and (20) a polypeptide, such as an hIL-1Ra1Vpolypeptide, consisting of amino acid residues from at or about 46 to ator about 218, inclusive of FIG. 19 (SEQ ID NO:25); or (b) the complementof the DNA of (a).

In another aspect, the invention provides an isolated DNA moleculeselected from the group consisting of: (1) a DNA molecule encoding apolypeptide, such as an hIL-1Ra1 polypeptide, comprising the amino acidsequence of amino acid residues from at or about 37 to at or about 203,inclusive of FIG. 2 (SEQ ID NO:5); (2) a DNA molecule encoding apolypeptide, such as an hIL-1Ra1 polypeptide, comprising the amino acidsequence of amino acid residues from at or about 15 to at or about 193,inclusive of FIG. 3 (SEQ ID NO:7); (3) a DNA molecule encoding apolypeptide, such as an hIL-1Ra2 polypeptide, comprising the amino acidsequence of amino acid residues from at or about 1 to at or about 134,inclusive of FIG. 5 (SEQ ID NO:10); (4) a DNA molecule encoding apolypeptide comprising the amino acid sequence of amino acid residuesfrom at or about 10 to at or about 134, inclusive of FIG. 5 (SEQ IDNO:10); (5) a DNA molecule encoding a polypeptide, such as an hIL-1Ra2fusion variant polypeptide, consisting of a native amino acid sequenceof hIL-1Ra2 consisting of amino acid residues from at or about 27 to ator about 134, inclusive of FIG. 5 (SEQ ID NO:10) fused at its N-terminusor C-terminus to a heterologous amino acid or amino acid sequence; (6) aDNA molecule encoding a polypeptide, such as an hIL-1Ra2 polypeptide,consisting of the amino acid sequence of amino acid residues from at orabout 27 to at or about 134, inclusive of FIG. 5 (SEQ ID NO:10); (7) aDNA molecule encoding a polypeptide, such as an hIL-1Ra3 polypeptide,comprising the amino acid sequence of amino acid residues from at orabout 95 to at or about 134, inclusive of FIG. 7 (SEQ ID NO:13); (8) aDNA molecule encoding a polypeptide, such as a mIL-1Ra3 polypeptide,comprising the amino acid sequence of amino acid residues from about 95to at or about 134, inclusive of FIG. 9 (SEQ ID NO:16); and (9) thecomplement of any of the DNA molecules of (1)-(8).

In another aspect, the invention provides an isolated DNA moleculeselected from the group consisting of: (1) a DNA molecule encoding apolypeptide, such as an hIL-1Ra1L polypeptide, comprising the amino acidsequence of amino acid residues from at or about 26 to at or about 207,inclusive of FIG. 15 (SEQ ID NO:19); (2) a DNA molecule encoding apolypeptide, such as an hIL-1Ra1S polypeptide, comprising the amino acidsequence of amino acid residues from at or about 26 to at or about 167,inclusive of FIG. 16 (SEQ ID NO:21); (3) a DNA molecule encoding apolypeptide, such as an hIL-1Ra1V polypeptide, comprising the amino acidsequence of amino acid residues from at or about 37 to at or about 218,inclusive of FIG. 19 (SEQ ID NO:25); (4) a DNA molecule encoding apolypeptide, such as an hIL-1Ra1V polypeptide, comprising the amino acidsequence of amino acid residues from at or about 46 to at or about 218,inclusive of FIG. 19 (SEQ ID NO:25); and (5) the complement of any ofthe DNA molecules of (1)-(4).

In another aspect, the invention provides an isolated DNA moleculeselected from the group consisting of: (1) a DNA molecule encoding apolypeptide, such as an hIL-1Ra1L polypeptide, comprising the amino acidsequence of amino acid residues from at or about 1 to at or about 207,inclusive of FIG. 15 (SEQ ID NO:19); (2) a DNA molecule encoding apolypeptide, such as an hIL-1Ra1S polypeptide, comprising the amino acidsequence of amino acid residues from at or about 1 to at or about 167,inclusive of FIG. 16 (SEQ ID NO:21); (3) a DNA molecule encoding apolypeptide, such as an hIL-1Ra1V polypeptide, comprising the amino acidsequence of amino acid residues from at or about 12 to at or about 218,inclusive of FIG. 19 (SEQ ID NO:25); (4) a DNA molecule encoding apolypeptide, such as an hIL-1Ra1V polypeptide, comprising the amino acidsequence of amino acid residues from at or about 1 to at or about 218,inclusive of FIG. 19 (SEQ ID NO:25); and (5) the complement of any ofthe DNA molecules of (1)-(4).

In another aspect, the invention provides an isolated DNA moleculeselected from the group consisting of: (1) a DNA molecule encoding apolypeptide, such as an hIL-1Ra3 polypeptide, comprising amino acidresidues from at or about 80 to at or about 155, inclusive of FIG. 7(SEQ ID NO:13); and (2) the complement of the DNA molecule of (1).

In another aspect, the invention provides an isolated DNA moleculeselected from the group consisting of: (1) a DNA molecule encoding apolypeptide, such as an hIL-1Ra1 polypeptide, comprising the amino acidsequence of amino acid residues from at or about 1 to at or about 203,inclusive of FIG. 2 (SEQ ID NO:5); (2) a DNA molecule encoding apolypeptide, such as an hIL-1Ra1 polypeptide, comprising the amino acidsequence of amino acid residues from at or about 1 to at or about 193,inclusive of FIG. 3 (SEQ ID NO:7); (3) a DNA molecule encoding apolypeptide, such as an hIL-1Ra3 polypeptide, comprising the amino acidsequence of amino acid residues from at or about 34 to at or about 155,inclusive of FIG. 7 (SEQ ID NO:13); (4) a DNA molecule encoding apolypeptide, such as a mIL-1Ra3 polypeptide, comprising the amino acidsequence of amino acid residues from at or about 34 to at or about 155,inclusive of FIG. 9 (SEQ ID NO:16); and (5) the complement of any of theDNA molecules of (1)-(4).

In another aspect, the invention provides an isolated DNA moleculeselected from the group consisting of: (1) a DNA molecule encoding apolypeptide, such as an hIL-1Ra3 polypeptide, comprising the amino acidsequence of amino acid residues from at or about 1 to at or about 155,inclusive of FIG. 7 (SEQ ID NO:13); (2) a DNA molecule encoding apolypeptide, such as a mIL-1Ra3 polypeptide, comprising the amino acidsequence of amino acid residues from at or about 1 to at or about 155,inclusive of FIG. 9 (SEQ ID NO:16); and (3) the complement of any of theDNA molecules of (1)-(2).

In another aspect, the invention provides an isolated DNA moleculeselected from the group consisting of: (1) a DNA molecule encoding apolypeptide, such as an hIL-1Ra3 polypeptide, comprising the amino acidsequence of amino acid residues from at or about 2 to at or about 155,inclusive of FIG. 7 (SEQ ID NO:13); (2) a DNA molecule encoding apolypeptide, such as a mIL-1Ra3 polypeptide, comprising the amino acidsequence of amino acid residues from at or about 2 to at or about 155,inclusive of FIG. 9 (SEQ ID NO:16); and (3) the complement of any of theDNA molecules of (1)-(2).

In another aspect, the invention provides an isolated nucleic acidmolecule comprising (a) a DNA molecule encoding a polypeptide selectedfrom the group consisting of: (1) a polypeptide comprising an hIL-1Ra1polypeptide, such as a mature hIL-1Ra1 polypeptide, encoded by the cDNAinsert in the vector deposited as ATCC Deposit No. 203588; (2) apolypeptide comprising an hIL-1Ra1 polypeptide, such as a maturehIL-1Ra1 polypeptide, encoded by the cDNA insert in the vector depositedas ATCC Deposit No. 203587; (3) a polypeptide consisting of an hIL-1Ra2polypeptide, such as a mature hIL-1Ra2 polypeptide, encoded by the cDNAinsert in the vector deposited as ATCC Deposit No. 203586, whichhIL-1Ra2 polypeptide is fused at its N-terminus or C-terminus to aheterologous amino acid or amino acid sequence; (4) a polypeptideconsisting of an hIL-1Ra2 polypeptide, such as a mature hIL-1Ra2polypeptide, encoded by the cDNA insert in the vector deposited as ATCCDeposit No. 203586; (5) a polypeptide comprising an hIL-1Ra3polypeptide, such as a mature hIL-1Ra3 polypeptide, encoded by the cDNAinsert in the vector deposited as ATCC Deposit No. 203589; and (6) apolypeptide comprising a mIL-1Ra3 polypeptide, such as a mature mIL-1Ra3polypeptide, encoded by the cDNA insert in the vector deposited as ATCCDeposit No. 203590; or (b) the complement of the DNA molecule of (a).

In another aspect, the invention provides an isolated nucleic acidmolecule comprising (a) a DNA molecule encoding a polypeptide selectedfrom the group consisting of: (1) a polypeptide comprising an hIL-1Ra1Lpolypeptide, such as a mature hIL-1Ra1L polypeptide, encoded by the cDNAinsert in the vector deposited as ATCC Deposit No. 203846; (2) apolypeptide consisting of an hIL-1Ra1S polypeptide, such as a maturehIL-1Ra1S polypeptide, encoded by the cDNA insert in the vectordeposited as ATCC Deposit No. 203855, which hIL-1Ra1S polypeptide isfused at its N-terminus or C-terminus to a heterologous amino acid oramino acid sequence; (3) a polypeptide consisting of an hIL-1Ra1Spolypeptide, such as a mature hIL-1Ra1S polypeptide, encoded by the cDNAinsert in the vector deposited as ATCC Deposit No. 203855; (4) apolypeptide comprising an hIL-1Ra1V polypeptide, such as a maturehIL-1Ra1V polypeptide, encoded by the cDNA insert in the vectordeposited as ATCC Deposit No. 203973; or (b) the complement of the DNAmolecule of (a).

In another aspect, the invention provides an isolated nucleic acidmolecule comprising (a) a DNA molecule encoding a polypeptide comprisingan hIL-1Ra1S polypeptide, such as a mature hIL-1Ra1S polypeptide,encoded by the cDNA insert in the vector deposited as ATCC Deposit No.203855; or (b) the complement of the DNA molecule of (a).

In another aspect, the invention provides an isolated nucleic acidmolecule comprising (a) a DNA encoding a polypeptide selected from thegroup consisting of: (1) a polypeptide comprising the entire amino acidsequence encoded by the longest open reading frame in the cDNA insert inthe vector deposited as ATCC Deposit No. 203588; (2) a polypeptidecomprising the entire amino acid sequence, or the entire amino acidsequence excluding the 36 N-terminal amino acid residues of suchsequence, encoded by the longest open reading frame in the cDNA insertin the vector deposited as ATCC Deposit No. 203587; (3) a polypeptidecomprising the entire amino acid sequence, or the entire amino acidsequence excluding the N-terminal amino acid residue of such sequence,or the entire amino acid sequence excluding the 9 N-terminal amino acidresidues of such sequence, encoded by the longest open reading frame inthe cDNA insert in the vector deposited as ATCC Deposit No. 203586; (4)a polypeptide comprising the entire amino acid sequence, or the entireamino acid sequence excluding the N-terminal amino acid residue of suchsequence, encoded by the longest open reading frame in the cDNA insertin the vector deposited as ATCC Deposit No. 203589; and (5) apolypeptide comprising the entire amino acid sequence, or the entireamino acid sequence excluding the N-terminal amino acid residue of suchsequence, encoded by the longest open reading frame in the cDNA insertin the vector deposited as ATCC Deposit No. 203590; or (b) thecomplement of the DNA of (a).

In another aspect, the invention provides an isolated nucleic acidmolecule comprising (a) a DNA encoding a polypeptide comprising theentire amino acid sequence encoded by the longest open reading frame inthe cDNA insert of a vector selected from the group consisting of thevectors deposited as ATCC Deposit Nos. 203588, 203586, 203589, 203590,and 203973, or (b) the complement of the DNA of (a).

In another aspect, the invention provides an isolated nucleic acidmolecule comprising (a) a DNA encoding a polypeptide selected from thegroup consisting of: (1) a polypeptide comprising the entire amino acidsequence, or the entire amino acid sequence excluding the N-terminalamino acid residue of such sequence, or the entire amino acid sequenceexcluding the 34 N-terminal amino acid residues of such sequence,encoded by the longest open reading frame in the cDNA insert in thevector deposited as ATCC Deposit No. 203846; (2) a polypeptidecomprising the entire amino acid sequence, or the entire amino acidsequence excluding the N-terminal amino acid residue of such sequence,or the entire amino acid sequence excluding the 25 N-terminal amino acidresidues of such sequence, encoded by the longest open reading frame inthe cDNA insert in the vector deposited as ATCC Deposit No. 204855; and(3) a polypeptide comprising the entire amino acid sequence, or theentire amino acid sequence excluding the N-terminal amino acid residueof such sequence, or the entire amino acid sequence excluding the 11N-terminal amino acid residues of such sequence, or the entire aminoacid sequence excluding the 36 N-terminal amino acid residues of suchsequence, or the entire amino acid sequence excluding the 45 N-terminalamino acid residues of such sequence, encoded by the longest openreading frame in the cDNA insert in the vector deposited as ATCC DepositNo. 203973; or (b) the complement of the DNA of (a).

In another aspect, the invention provides an isolated nucleic acidmolecule comprising (a) a DNA encoding a non-naturally occurring,chimeric polypeptide formed by fusing the entire amino acid sequenceexcluding the 38 N-terminal amino acid residues of such sequence, or theentire amino acid sequence excluding the 46 N-terminal amino acidresidues of such sequence, encoded by the longest open reading frame inthe cDNA insert in the vector deposited as ATCC Deposit No. 203855, atits N-terminus or C-terminus to a heterologous amino acid or amino acidsequence; or (b) the complement of the DNA of (a).

In another aspect, the invention provides an isolated nucleic acidmolecule comprising (a) a DNA encoding a polypeptide consisting of theentire amino acid sequence excluding the 38 N-terminal amino acidresidues of such sequence, or the entire amino acid sequence excludingthe 46 N-terminal amino acid residues of such sequence, encoded by thelongest open reading frame in the cDNA insert in the vector deposited asATCC Deposit No. 203855; or (b) the complement of the DNA of (a).

In another aspect, the invention provides an isolated nucleic acidmolecule comprising (a) a DNA molecule encoding a polypeptide comprisingthe entire amino acid sequence encoded by the longest open reading framein the cDNA insert of a vector selected from the group consisting of thevectors deposited as ATCC Deposit Nos. 203846, 203855 and 203973, or (b)the complement of the DNA of (a).

In another aspect, the invention provides an isolated DNA moleculeselected from the group consisting of: (1) a DNA molecule which encodesa polypeptide, such as an hIL-1Ra1V fusion variant polypeptide,consisting of a native amino acid sequence of hIL-1Ra1V consisting ofamino acid residues from at or about 37 to at or about 218, inclusive ofFIG. 19 (SEQ ID NO:25) fused at its N-terminus or C-terminus to aheterologous amino acid or amino acid sequence, and which DNA moleculecomprises the nucleic acid sequence in the sense strand of FIG. 19 (SEQID NO:24) that encodes the native amino acid sequence; (2) a DNAmolecule which encodes a polypeptide, such as an hIL-1Ra1V fusionvariant polypeptide, consisting of a native amino acid sequence ofhIL-1Ra1V consisting of amino acid residues from at or about 46 to at orabout 218, inclusive of FIG. 19 (SEQ ID NO:25) fused at its N-terminusor C-terminus to a heterologous amino acid or amino acid sequence, andwhich DNA molecule comprises the nucleic acid sequence in the sensestrand of FIG. 19 (SEQ ID NO:24) that encodes the native amino acidsequence; (3) a DNA molecule which encodes a polypeptide, such as anhIL-1Ra1V polypeptide, consisting of a native amino acid sequence ofhIL-1Ra1V consisting of amino acid residues from at or about 37 to at orabout 218, inclusive of FIG. 19 (SEQ ID NO:25), and which DNA moleculecomprises the nucleic acid sequence in the sense strand of FIG. 19 (SEQID NO:24) that encodes the native amino acid sequence; (4) a DNAmolecule which encodes a polypeptide, such as an hIL-1Ra1V polypeptide,consisting of a native amino acid sequence of hIL-1Ra1V consisting ofamino acid residues from at or about 46 to at or about 218, inclusive ofFIG. 19 (SEQ ID NO:25), and which DNA molecule comprises the nucleicacid sequence in the sense strand of FIG. 19 (SEQ ID NO:24) that encodesthe native amino acid sequence; and (5) the complement of any of the DNAmolecules of (1)-(4).

In another aspect, the invention provides an isolated DNA moleculeselected from the group consisting of: (1) a DNA molecule which encodesa polypeptide, such as an hIL-1Ra1 polypeptide, and which DNA moleculecomprises the nucleic acid sequence of nucleotide positions from at orabout 118 to at or about 618, inclusive in the sense strand of FIG. 2(SEQ ID NO:4); (2) a DNA molecule which encodes a polypeptide, such asan hIL-1Ra1 polypeptide, and which DNA molecule comprises the nucleicacid sequence of nucleotide positions from at or about 145 to at orabout 681, inclusive in the sense strand of FIG. 3 (SEQ ID NO:6); (3) aDNA molecule which encodes a polypeptide, such as an hIL-1Ra2polypeptide, and which DNA molecule comprises the nucleic acid sequenceof nucleotide positions from at or about 96 to at or about 497,inclusive in the sense strand of FIG. 5 (SEQ ID NO:9); (4) a DNAmolecule which comprises the nucleic acid sequence of nucleotidepositions from at or about 123 to at or about 497, inclusive in thesense strand of FIG. 5 (SEQ ID NO:9); (5) a DNA molecule which encodes apolypeptide, such as an hIL-1Ra2 fusion variant polypeptide, consistingof a native amino acid sequence of hIL-1Ra2 consisting of amino acidresidues from at or about 27 to at or about 134, inclusive of FIG. 5(SEQ ID NO:10) fused at its N-terminus or C-terminus to a heterologousamino acid or amino acid sequence, and which DNA molecule comprises thenucleic acid sequence in the sense strand of FIG. 5 (SEQ ID NO:9) thatencodes the native amino acid sequence; (6) a DNA molecule which encodesa polypeptide, such as an hIL-1Ra2 polypeptide, consisting of a nativeamino acid sequence of hIL-1Ra2 consisting of amino acid residues fromat or about 27 to at or about 134, inclusive of FIG. 5 (SEQ ID NO:10),and which DNA molecule comprises the nucleic acid sequence in the sensestrand of FIG. 5 (SEQ ID NO:9) that encodes the native amino acidsequence; (7) a DNA molecule which encodes a polypeptide, such as anhIL-1Ra3 polypeptide, and which DNA molecule comprises the nucleic acidsequence of nucleotide positions from at or about 283 to at or about402, inclusive in the sense strand of FIG. 7 (SEQ ID NO:12); (8) a DNAmolecule which encodes a polypeptide, such as a mIL-1Ra3 polypeptide,and which DNA molecule comprises the nucleic acid sequence of nucleotidepositions from at or about 427 to at or about 546, inclusive in thesense strand of FIG. 9 (SEQ ID NO:15); and (9) the complement of any ofthe DNA molecules of (1)-(8).

In another aspect, the invention provides an isolated DNA moleculeselected from the group consisting of: (1) a DNA molecule which encodesa polypeptide, such as an hIL-1Ra1L polypeptide, and which DNA moleculecomprises the nucleic acid sequence of nucleotide positions from at orabout 79 to at or about 624, inclusive in the sense strand of FIG. 15(SEQ ID NO:18); (2) a DNA molecule which encodes a polypeptide, such asan hIL-1Ra1S polypeptide, and which DNA molecule comprises the nucleicacid sequence of nucleotide positions from at or about 79 to at or about504, inclusive in the sense strand of FIG. 16 (SEQ ID NO:20); (3) a DNAmolecule which encodes a polypeptide, such as an hIL-1Ra1S fusionvariant polypeptide, consisting of a native amino acid sequence ofhIL-1Ra1S consisting of amino acid residues from at or about 39 to at orabout 167, inclusive of FIG. 16 (SEQ ID NO:21) fused at its N-terminusor C-terminus to a heterologous amino acid or amino acid sequence, andwhich DNA molecule comprises the nucleic acid sequence in the sensestrand of FIG. 16 (SEQ ID NO:20) that encodes the native amino acidsequence; (4) a DNA molecule which encodes a polypeptide, such as anhIL-1Ra1S fusion variant polypeptide, consisting of a native amino acidsequence of hIL-1Ra1S consisting of amino acid residues from at or about47 to at or about 167, inclusive of FIG. 16 (SEQ ID NO:21) fused at itsN-terminus or C-terminus to a heterologous amino acid or amino acidsequence, and which DNA molecule comprises the nucleic acid sequence inthe sense strand of FIG. 16 (SEQ ID NO:20) that encodes the native aminoacid sequence; (5) a DNA molecule which encodes a polypeptide, such asan hIL-1Ra1S polypeptide, consisting of a native amino acid sequence ofhIL-1Ra1S consisting of amino acid residues from at or about 39 to at orabout 167, inclusive of FIG. 16 (SEQ ID NO:21), and which DNA moleculecomprises the nucleic acid sequence in the sense strand of FIG. 16 (SEQID NO:20) that encodes the native amino acid sequence; (6) a DNAmolecule which encodes a polypeptide, such as an hIL-1Ra1S polypeptide,consisting of a native amino acid sequence of hIL-1Ra1S consisting ofamino acid residues from at or about 47 to at or about 167, inclusive ofFIG. 16 (SEQ ID NO:21), and which DNA molecule comprises the nucleicacid sequence in the sense strand of FIG. 16 (SEQ ID NO:20) that encodesthe native amino acid sequence; (7) a DNA molecule which encodes apolypeptide, such as an hIL-1Ra1V polypeptide, and which DNA moleculecomprises the nucleic acid sequence of nucleotide positions from at orabout 181 to at or about 729, inclusive in the sense strand of FIG. 19(SEQ ID NO:24); (8) a DNA molecule which encodes a polypeptide, such asan hIL-1Ra1V polypeptide, and which DNA molecule comprises the nucleicacid sequence of nucleotide positions from at or about 208 to at orabout 729, inclusive in the sense strand of FIG. 19 (SEQ ID NO:24); and(9) the complement of any of the DNA molecules of (1)-(8).

In another aspect, the invention provides an isolated DNA moleculeselected from the group consisting of: (1) a DNA molecule which encodesa polypeptide, such as an hIL-1Ra1L polypeptide, and which DNA moleculecomprises the nucleic acid sequence of nucleotide positions from at orabout 4 to at or about 624, inclusive in the sense strand of FIG. 15(SEQ ID NO:18); (2) a DNA molecule which encodes a polypeptide, such asan hIL-1Ra1S polypeptide, and which DNA molecule comprises the nucleicacid sequence of nucleotide positions from at or about 4 to at or about504, inclusive in the sense strand of FIG. 16 (SEQ ID NO:20); (3) a DNAmolecule which encodes a polypeptide, such as an hIL-1Ra1V polypeptide,and which DNA molecule comprises the nucleic acid sequence of nucleotidepositions from at or about 106 to at or about 729, inclusive in thesense strand of FIG. 19 (SEQ ID NO:24); (4) a DNA molecule which encodesa polypeptide, such as an hIL-1Ra1V polypeptide, and which comprises thenucleic acid sequence of nucleotide positions from at or about 73 to ator about 729, inclusive in the sense strand of FIG. 19 (SEQ ID NO:24);and (5) the complement of any of the DNA molecules of (1)-(4).

In another aspect, the invention provides an isolated DNA moleculeselected from the group consisting of: (1) a DNA molecule comprising thenucleic acid sequence of nucleotide positions from at or about 103 to ator about 681, inclusive in the sense strand of FIG. 3 (SEQ ID NO:6); (2)a DNA molecule comprising the nucleic acid sequence of nucleotidepositions from at or about 100 to at or about 465, inclusive in thesense strand of FIG. 7 (SEQ ID NO:12); (3) a DNA molecule comprising thenucleic acid sequence of nucleotide positions from at or about 244 to ator about 609, inclusive in the sense strand of FIG. 9 (SEQ ID NO:15);and (4) the complement of any of the DNA molecules of (1)-(3).

In another aspect, the invention provides an isolated DNA moleculecomprising (a) the complete DNA sequence in the sense strand of FIG. 2(SEQ ID NO:4), FIG. 3 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:9), FIG. 7 (SEQID NO:12), or FIG. 9 (SEQ ID NO:15), or (b) the complement of (a).

In another aspect, the invention provides an isolated DNA moleculecomprising (a) the complete DNA sequence in the sense strand of FIG. 15(SEQ ID NO:18), FIG. 16 (SEQ ID NO:20), or FIG. 19 (SEQ ID NO:24), or(b) the complement of (a).

In a specific aspect, the invention provides an isolated nucleic acidmolecule comprising DNA encoding an IL-1lp polypeptide, with or withoutthe N-terminal signal sequence and/or the initiating methionine, or iscomplementary to such IL-1lp encoding nucleic acid molecule. The signalpeptide has been tentatively identified as extending from amino acidposition 1 to at or about amino acid position 14 in the IL-1lp sequenceof FIG. 3 (SEQ ID NO:7), from amino acid position 1 to at or about aminoacid position 26 in the IL-1lp sequence of FIG. 5 (SEQ ID NO:10), fromamino acid position 1 to at or about amino acid position 33 in theIL-1lp sequence of FIG. 7 (SEQ ID NO:13), and from amino acid position 1to at or about amino acid position 33 in the IL-1lp sequence of FIG. 9(SEQ ID NO:16).

The IL-1lp sequence of amino acids from at or about 1 to at or about 207of FIG. 15 (SEQ ID NO:19) is believed to behave as a mature sequence(without a presequence that is removed in post-translational processing)in certain animal cells. In addition, it is believed that other animalcells recognize and remove in post-translational processing one or moresignal peptide(s) contained in the sequence of amino acid positions 1 toabout 34 of FIG. 15 (SEQ ID NO:19).

The IL-1lp sequence of amino acids from at or about 1 to at or about 167of FIG. 16 (SEQ ID NO:21) is believed to behave as a mature sequence(without a presequence that is removed in post-translational processing)in certain animal cells. In addition, it is believed that other animalcells recognize and remove in post-translational processing one or moresignal peptide(s) contained in the sequence of amino acid positions 1 toabout 46 in the IL-1lp sequence of FIG. 16 (SEQ ID NO:21).

The IL-1lp sequence of amino acids from at or about 1 to at or about 218of FIG. 19 (SEQ ID NO:25) is believed to behave as a mature sequence(without a presequence that is removable in post-translationalprocessing) in certain animal cells. The IL-1lp sequence of amino acidsfrom at or about 12 to at or about 218 of FIG. 19 (SEQ ID NO:25) thatresults from initiation of translation at the start codon occurring atnucleotide positions 106-108 is also believed to behave as maturesequence in certain animal cells. It is further believed that otheranimal cells recognize and remove in post-translational processing oneor more signal peptide(s) contained in the sequence of amino acidpositions 1 to 45 in the IL-1lp polypeptide of amino acid positions 1 to218 of FIG. 19 (SEQ ID NO:25) or contained in the sequence of amino acidpositions 12 to 45 in the IL-1lp polypeptide of amino acid positions 12to 218 of FIG. 19 (SEQ ID NO:25).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide thatretains at least one biologic activity of a native sequence IL-1lp, suchas the IL-18R binding activity of a native sequence hIL-1Ra1, or theIL-1R binding activity of a native sequence hIL-1Ra3 or mIL-1Ra3, andwhich IL-1lp polypeptide has at least at or about 80% positives, or atleast at or about 85% positives, or at least at or about 90% positives,or at least at or about 95% positives to an amino acid sequence selectedfrom the group consisting of: (1) amino acid residues from at or about37 to at or about 203, inclusive of FIG. 2 (SEQ ID NO:5), (2) amino acidresidues from at or about 15 to at or about 193, inclusive of FIG. 3(SEQ ID NO:7), (3) amino acid residues from at or about 34 to at orabout 155, inclusive of FIG. 7 (SEQ ID NO:13), (4) amino acid residuesfrom at or about 34 to at or about 155, inclusive of FIG. 9 (SEQ IDNO:16), (5) amino acid residues from at or about 26 to at or about 207,inclusive of FIG. 15 (SEQ ID NO:19), and (6) amino acid residues from ator about 46 to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25), or(b) the complement of the DNA of (a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide thatretains at least one biologic activity of a native sequence IL-1lp, suchas the IL-18R binding activity of a native sequence hIL-1Ra1, or theIL-1R binding activity of a native sequence hIL-1Ra3 or mIL-1Ra3, andwhich IL-1lp polypeptide has at least at or about 80% positives, or atleast at or about 85% positives, or at least at or about 90% positives,or at least at or about 95% positives to an amino acid sequence selectedfrom the group consisting of: (1) amino acid residues from at or about95 to at or about 134, inclusive of FIG. 7 (SEQ ID NO:13), and (2) aminoacid residues from at or about 95 to at or about 134, inclusive of FIG.9 (SEQ ID NO:16), or (b) the complement of the DNA of (a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide thatretains at least one biologic activity of a native sequence IL-1lp, suchas the IL-18R binding activity of a native sequence hIL-Ra1, or theIL-1R binding activity of a native sequence hIL-1Ra3 or mIL-1Ra3, andwhich IL-1lp polypeptide has at least at or about 80% positives, or atleast at or about 85% positives, or at least at or about 90% positives,or at least at or about 95% positives to the amino acid sequence ofamino acid residues from at or about 80 to at or about 155, inclusive ofFIG. 7 (SEQ ID NO:13), or (b) the complement of the DNA of (a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide thatretains at least one biologic activity of a native sequence IL-1lp, suchas the IL-18R binding activity of a native sequence hIL-1Ra1, or theIL-1R binding activity of a native sequence hIL-1Ra3 or mIL-1Ra3, andwhich IL-1lp polypeptide has at least at or about 80% positives, or atleast at or about 85% positives, or at least at or about 90% positives,or at least at or about 95% positives to an amino acid sequence selectedfrom the group consisting of: (1) amino acid residues from at or about 2to at or about 155, inclusive of FIG. 7 (SEQ ID NO:13), and (2) aminoacid residues from at or about 2 to at or about 155, inclusive of FIG. 9(SEQ ID NO:16), or (b) the complement of the DNA of (a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide havingat least at or about 80% positives, or at least at or about 85%positives, or at least at or about 90% positives, or at least at orabout 95% positives to an amino acid sequence selected from the groupconsisting of: (1) amino acid residues from at or about 95 to at orabout 134, inclusive of FIG. 7 (SEQ ID NO:13), and (2) amino acidresidues from at or about 95 to at or about 134, inclusive of FIG. 9(SEQ ID NO:16), or (b) the complement of the DNA of (a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide havingat least at or about 80% positives, or at least at or about 85%positives, or at least at or about 90% positives, or at least at orabout 95% positives to the amino acid sequence of amino acid residuesfrom at or about 80 to at or about 155, inclusive of FIG. 7 (SEQ IDNO:13), or (b) the complement of the DNA of (a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide havingat least at or about 80% positives, or at least at or about 85%positives, or at least at or about 90% positives, or at least at orabout 95% positives to an amino acid sequence selected from the groupconsisting of: (1) amino acid residues from at or about 2 to at or about155, inclusive of FIG. 7 (SEQ ID NO:13), and (2) amino acid residuesfrom at or about 2 to at or about 155, inclusive of FIG. 9 (SEQ IDNO:16), or (b) the complement of the DNA of (a).

In a still further aspect, the invention concerns an isolated nucleicacid molecule comprising (a) DNA encoding an IL-1lp polypeptide thatretains at least one biologic activity of a native sequence IL-1lp, suchas the IL-18R binding activity of a native sequence hIL-1Ra1, hIL-1Ra1L,or hIL-1Ra1V, and which IL-1lp polypeptide has at least at or about 80%positives, or at least at or about 85% positives, or at least at orabout 90% positives, or at least at or about 95% positives to an aminoacid sequence selected from the group consisting of: (1) amino acidresidues from at or about 1 to at or about 207, inclusive of FIG. 15(SEQ ID NO:19), and (2) amino acid residues from at or about 1 to at orabout 218, inclusive of FIG. 19 (SEQ ID NO:25), or (b) the complement ofthe DNA of (a).

In another embodiment, the invention provides a vector comprising DNAencoding IL-1lp or its variants. The vector may comprise any of theisolated nucleic acid molecules hereinabove defined.

A host cell comprising such a vector is also provided. By way ofexample, the host cells may be CHO cells, E. coli, or yeast. A processfor producing IL-1lp polypeptides is further provided and comprisesculturing host cells under conditions suitable for expression of IL-1lpand recovering IL-1lp from the cell culture.

In another embodiment, the invention provides isolated IL-1lppolypeptide encoded by any of the isolated nucleic acid sequenceshereinabove defined.

In another aspect, the invention provides isolated native sequenceIL-1lp polypeptide, which in one embodiment, comprises an amino acidsequence selected from the group consisting of: (1) the amino acidsequence of residues from at or about 37 to at or about 203, inclusiveof FIG. 2 (SEQ ID NO:5), (2) the amino acid sequence of residues from ator about 15 to at or about 193, inclusive of FIG. 3 (SEQ ID NO:7), (3)the amino acid sequence of residues from at or about 34 to at or about155, inclusive of FIG. 7 (SEQ ID NO:13), and (4) the amino acid sequenceof residues from at or about 34 to at or about 155, inclusive of FIG. 9(SEQ ID NO:16).

In another aspect, the invention provides isolated native sequenceIL-1lp polypeptide, which in one embodiment, comprises an amino acidsequence selected from the group consisting of: (1) the amino acidsequence of residues from at or about 1 to at or about 207, inclusive ofFIG. 15 (SEQ ID NO:19), (2) the amino acid sequence of residues from ator about 26 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19), (3)the amino acid sequence of residues from at or about 1 to at or about167, inclusive of FIG. 16 (SEQ ID NO:21), (4) the amino acid sequence ofresidues from at or about 26 to at or about 167, inclusive of FIG. 16(SEQ ID NO:21), (5) the amino acid sequence of residues from at or about1 to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25), (6) the aminoacid sequence of residues from at or about 12 to at or about 218,inclusive of FIG. 19 (SEQ ID NO:25), (7) the amino acid sequence ofresidues from at or about 37 to at or about 218, inclusive of FIG. 19(SEQ ID NO:25), and (8) the amino acid sequence of residues from at orabout 46 to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25).

In another aspect, the invention concerns an isolated IL-1lp polypeptidethat retains at least one biologic activity of a native sequence IL-1lp,such as the IL-1R binding activity of a native sequence hIL-1Ra3 ormIL-1Ra3, or the IL18R binding activity of a native sequence hIL-1Ra1,hIL-1Ra1L, or hIL-1Ra1V, and that consists of an amino acid sequencehaving at least at or about 80% sequence identity, or at least at orabout 85% sequence identity, or at least at or about 90% sequenceidentity, or at least at or about 95% sequence identity to the sequenceof amino acid residues from at or about 37 to at or about 203, inclusiveof FIG. 2 (SEQ ID NO:5), the sequence of amino acid residues from at orabout 15 to at or about 193, inclusive of FIG. 3 (SEQ ID NO:7), thesequence of amino acid residues from at or about 34 to at or about 155,inclusive of FIG. 7 (SEQ ID NO:13), the sequence of amino acid residuesfrom at or about 34 to at or about 155, inclusive of FIG. 9 (SEQ IDNO:16), the sequence of amino acid residues from at or about 26 to at orabout 207, inclusive of FIG. 15 (SEQ ID NO:19), or the sequence of aminoacid residues from at or about 46 to at or about 218, inclusive of FIG.19 (SEQ ID NO:25).

In another aspect, the invention concerns an isolated IL-1lp polypeptidethat retains at least one biologic activity of a native sequence IL-1lp,such as the IL-18R binding activity of a native sequence hIL-1Ra1,hIL-1Ra1L, or hIL-1Ra1V, and that consists of an amino acid sequencehaving at least at or about 80% sequence identity, or at least at orabout 85% sequence identity, or at least at or about 90% sequenceidentity, or at least at or about 95% sequence identity to the sequenceof amino acid residues from at or about 1 to at or about 207, inclusiveof FIG. 15 (SEQ ID NO:19), or the sequence of amino acid residues fromat or about 1 to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25).

In another aspect, the invention provides an isolated IL-1lp selectedfrom the group consisting of: (1) a polypeptide, such as an hIL-1Ra1polypeptide, consisting of an amino acid sequence having a sequenceidentity of at least at or about 80%, or at least at or about 85%, or atleast at or about 90%, or at least at or about 95%, to the sequence ofamino acid residues from at or about 37 to at or about 63, inclusive ofFIG. 2 (SEQ ID NO:5); (2) a polypeptide, such as an hIL-1Ra1polypeptide, consisting of an amino acid sequence having a sequenceidentity of at least at or about 80%, or at least at or about 85%, or atleast at or about 90%, or at least at or about 95%, to the sequence ofamino acid residues from at or about 15 to at or about 53, inclusive ofFIG. 3 (SEQ ID NO:7); (3) a polypeptide, such as an hIL-1Ra2polypeptide, comprising the amino acid sequence of amino acid residuesfrom at or about 1 to at or about 134, inclusive of FIG. 5 (SEQ IDNO:10); (4) a polypeptide comprising the amino acid sequence of aminoacid residues from at or about 10 to at or about 134, inclusive of FIG.5 (SEQ ID NO:10); (5) a polypeptide, such as an hIL-1Ra2 polypeptide,consisting of the amino acid sequence of amino acid residues from at orabout 27 to at or about 134, inclusive of FIG. 5 (SEQ ID NO:10); (6) apolypeptide, such as an hIL-1Ra2 fusion variant polypeptide, consistingof a native amino acid sequence of hIL-1Ra2 consisting of amino acidresidues from at or about 27 to at or about 134, inclusive of FIG. 5(SEQ ID NO:10) fused at its N-terminus or C-terminus to a heterologousamino acid or amino acid sequence; (7) a polypeptide, such as anhIL-1Ra3 polypeptide, consisting of an amino acid sequence having asequence identity of at least at or about 80%, or at least at or about85%, or at least at or about 90%, or at least at or about 95%, to thesequence of amino acid residues from at or about 95 to at or about 134,inclusive of FIG. 7 (SEQ ID NO:13); and (8) a polypeptide, such as amIL-1Ra3 polypeptide, consisting of an amino acid sequence having asequence identity of at least at or about 80%, or at least at or about85%, or at least at or about 90%, or at least at or about 95%, to thesequence of amino acid residues from at or about 95 to at or about 134,inclusive of FIG. 9 (SEQ ID NO:16).

In another aspect, the invention provides an isolated IL-1lp selectedfrom the group consisting of: (1) a polypeptide, such as an hIL-1Ra1Lpolypeptide, consisting of an amino acid sequence having a sequenceidentity of at least at or about 80%, or at least at or about 85%, or atleast at or about 90%, or at least at or about 95%, to the sequence ofamino acid residues from at or about 27 to at or about 44, inclusive ofFIG. 15 (SEQ ID NO:19); (2) a polypeptide, such as an hIL-1Ra1S fusionvariant polypeptide, consisting of a native amino acid sequence ofhIL-1Ra1S consisting of amino acid residues from at or about 39 to at orabout 167, inclusive of FIG. 16 (SEQ ID NO:21) fused at its N-terminusor C-terminus to a heterologous amino acid or amino acid sequence; (3) apolypeptide, such as an hIL-1Ra1S fusion variant polypeptide, consistingof a native amino acid sequence of h[L-1Ra1S consisting of amino acidresidues from at or about 47 to at or about 167, inclusive of FIG. 16(SEQ ID NO:21) fused at its N-terminus or C-terminus to a heterologousamino acid or amino acid sequence; (4) a polypeptide, such as anhIL-1Ra1S polypeptide, consisting of amino acid residues from at orabout 39 to at or about 167, inclusive of FIG. 16 (SEQ ID NO:21); (5) apolypeptide, such as an hIL-1Ra1S polypeptide, consisting of amino acidresidues from at or about 47 to at or about 167, inclusive of FIG. 16(SEQ ID NO:21); and (6) a polypeptide, such as an hIL-1Ra1V polypeptide,consisting of an amino acid sequence having a sequence identity of atleast at or about 80%, or at least at or about 85%, or at least at orabout 90%, or at least at or about 95%, to the sequence of amino acidresidues from at or about 37 to at or about 55, inclusive of FIG. 19(SEQ ID NO:25).

In another aspect, the invention concerns an isolated IL-1lp polypeptidethat consists of an amino acid sequence having at least at or about 80%sequence identity, or at least at or about 85% sequence identity, or atleast at or about 90% sequence identity, or at least at or about 95%sequence identity to the sequence of amino acid residues from at orabout 37 to at or about 203, inclusive of FIG. 2 (SEQ ID NO:5), thesequence of amino acid residues from at or about 15 to at or about 193,inclusive of FIG. 3 (SEQ ID NO:7), the sequence of amino acid residuesfrom at or about 34 to at or about 155, inclusive of FIG. 7 (SEQ IDNO:13), the sequence of amino acid residues from at or about 34 to at orabout 155, inclusive of FIG. 9 (SEQ ID NO:16), the sequence of aminoacid residues from at or about 26 to at or about 207, inclusive of FIG.15 (SEQ ID NO:19), or the sequence of amino acid residues from at orabout 46 to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25).

In another aspect, the invention concerns an isolated IL-1lp polypeptidethat consists of an amino acid sequence having at least at or about 80%sequence identity, or at least at or about 85% sequence identity, or atleast at or about 90% sequence identity, or at least at or about 95%sequence identity to the sequence of amino acid residues from at orabout 1 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19), or thesequence of amino acid residues from at or about 1 to at or about 218,inclusive of FIG. 19 (SEQ ID NO:25).

In another aspect, the invention provides an isolated polypeptide, suchas an hIL-1Ra3 polypeptide, consisting of an amino acid sequence havinga sequence identity of at least at or about 80%, or at least at or about85%, or at least at or about 90%, or at least at or about 95%, to thesequence of amino acid residues from at or about 80 to at or about 155,inclusive of FIG. 7 (SEQ ID NO:13).

In a further aspect, the invention concerns an isolated IL-1lppolypeptide that retains at least one biologic activity of a nativesequence IL-1lp, such as the IL-1R binding activity of a native sequencehIL-1Ra3 or mIL-1Ra3, or the IL-18R binding activity of a nativesequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, and that consists of anamino acid sequence having at least at or about 80% sequence identity,or at least at or about 85% sequence identity, or at least at or about90% sequence identity, or at least at or about 95% sequence identity tothe amino acid sequence of an IL-1lp, such as a mature IL-1lppolypeptide, encoded by the cDNA insert in the vector deposited as ATCCDeposit No. 203588 (DNA85066-2534), ATCC Deposit No. 203587(DNA96786-2534), ATCC Deposit No. 203589 (DNA96787-2534), ATCC DepositNo. 203590 (DNA92505-2534), ATCC Deposit No. 203846 (DNA 102043-2534),or ATCC Deposit No. 203973 (DNA114876-2534). In a preferred embodiment,the IL-1lp polypeptide comprises the amino acid sequence of an IL-1lp,such as a mature IL-1lp polypeptide, encoded by the cDNA insert in thevector deposited as ATCC Deposit No. 203588 (DNA85066-2534), ATCCDeposit No. 203587 (DNA96786-2534), ATCC Deposit No. 203586(DNA92929-2534), ATCC Deposit No. 203589 (DNA96787-2534), ATCC DepositNo. 203590 (DNA92505-2534), ATCC Deposit No. 203846 (DNA102043-2534),ATCC Deposit No. 203973 (DNA1 14876-2534), or ATCC Deposit No. 203855(DNA102044-2534).

In a further aspect, the invention concerns an isolated IL-1lppolypeptide that retains at least one biologic activity of a nativesequence IL-1lp, such as the IL-1R binding activity of a native sequencehIL-1Ra3 or mIL-1Ra3, or the IL-18R binding activity of a nativesequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, and that consists of anamino acid sequence having at least at or about 80% sequence identity,or at least at or about 85% sequence identity, or at least at or about90% sequence identity, or at least at or about 95% sequence identity tothe entire amino acid sequence encoded by the longest open reading framein the cDNA insert of a vector selected from the group consisting of thevectors deposited as ATCC Deposit No. 203588 (DNA85066-2534), ATCCDeposit No. 203587 (DNA96786-2534), ATCC Deposit No. 203589(DNA96787-2534), ATCC Deposit No. 203590 (DNA92505-2534), ATCC DepositNo. 203846 (DNA102043-2534), and ATCC Deposit No. 203973(DNA114876-2534). In a preferred embodiment, the IL-1lp polypeptidecomprises the entire amino acid sequence encoded by the longest openreading frame in the cDNA insert of a vector selected from the groupconsisting of the vectors deposited as ATCC Deposit No. 203588(DNA85066-2534), ATCC Deposit No. 203587 (DNA96786-2534), ATCC DepositNo. 203586 (DNA92929-2534), ATCC Deposit No. 203589 (DNA96787-2534), andATCC Deposit No. 203590 (DNA92505-2534). In another preferredembodiment, the IL-1lp polypeptide comprises the entire amino acidsequence encoded by the longest open reading frame in the cDNA insert ofa vector selected from the group consisting of the vectors deposited asATCC Deposit No. 203846 (DNA102043-2534), ATCC Deposit No. 203855(DNA102044-2534), and ATCC Deposit No. 203973 (DNA114876-2534).

In another aspect, the invention concerns an isolated IL-1lp polypeptidethat retains at least one biologic activity of a native sequence IL-1lp,such as the IL-1R binding activity of a native sequence IL-1Ra3 ormIL-1Ra3, or the IL-18R binding activity of a native sequence hIL-1Ra1,hIL-1Ra1L, or hIL-1Ra1V, and that consists of an amino acid sequencehaving at least at or about 80% sequence identity, or at least at orabout 85% sequence identity, or at least at or about 90% sequenceidentity, or at least at or about 95% sequence identity to an amino acidsequence selected from the group consisting of: (1) the entire aminoacid sequence encoded by the longest open reading frame in the cDNAinsert in the vector deposited as ATCC Deposit No. 203588, (2) theentire amino acid sequence, or the entire amino acid sequence excludingthe 36 N-terminal amino acid residues of such sequence, encoded by thelongest open reading frame in the cDNA insert in the vector deposited asATCC Deposit No. 203587, (3) the entire amino acid sequence, or theentire amino acid sequence excluding the N-terminal amino acid residueof such sequence, encoded by the longest open reading frame in the cDNAinsert in the vector deposited as ATCC Deposit No. 203589, (4) theentire amino acid sequence, or the entire amino acid sequence excludingthe N-terminal amino acid residue of such sequence, encoded by thelongest open reading frame in the cDNA insert in the vector deposited asATCC Deposit No. 203590, (5) the entire amino acid sequence, or theentire amino acid sequence excluding the N-terminal amino acid residueof such sequence, or the entire amino acid sequence excluding the 34N-terminal amino acid residues of such sequence, encoded by the longestopen reading frame in the cDNA insert in the vector deposited as ATCCDeposit No. 203846, and (6) the entire amino acid sequence, or theentire amino acid sequence excluding the N-terminal amino acid residueof such sequence, or the entire amino acid sequence excluding the 45N-terminal amino acid residues of such sequence, encoded by the longestopen reading frame in the cDNA insert in the vector deposited as ATCCDeposit No. 203973.

In a further aspect, the invention concerns an isolated IL-1lppolypeptide that consists of an amino acid sequence having at least ator about 80% sequence identity, or at least at or about 85% sequenceidentity, or at least at or about 90% sequence identity, or at least ator about 95% sequence identity to the amino acid sequence of an IL-1lp,such as a mature IL-1lp polypeptide, encoded by the cDNA insert in thevector deposited as ATCC Deposit No. 203588 (DNA85066-2534), ATCCDeposit No. 203587 (DNA96786-2534), ATCC Deposit No. 203589(DNA96787-2534), ATCC Deposit No. 203590 (DNA92505-2534), ATCC DepositNo. 203846 (DNA102043-2534), or ATCC Deposit No. 203973(DNA114876-2534).

In a further aspect, the invention concerns an isolated IL-1lppolypeptide that consists of an amino acid sequence having at least ator about 80% sequence identity, or at least at or about 85% sequenceidentity, or at least at or about 90% sequence identity, or at least ator about 95% sequence identity to the entire amino acid sequence encodedby the longest open reading frame in the cDNA insert of a vectorselected from the group consisting of the vectors deposited as ATCCDeposit No. 203588 (DNA85066-2534), ATCC Deposit No. 203587(DNA96786-2534), ATCC Deposit No. 203589 (DNA96787-2534), ATCC DepositNo. 203590 (DNA92505-2534), ATCC Deposit No. 203846 (DNA 102043-2534),and ATCC Deposit No. 203973 (DNA114876-2534).

In another aspect, the invention concerns an isolated IL-1lp polypeptidethat consists of an amino acid sequence having at least at or about 80%sequence identity, or at least at or about 85% sequence identity, or atleast at or about 90% sequence identity, or at least at or about 95%sequence identity to an amino acid sequence selected from the groupconsisting of: (1) the entire amino acid sequence encoded by the longestopen reading frame in the cDNA insert in the vector deposited as ATCCDeposit No. 203588, (2) the entire amino acid sequence, or the entireamino acid sequence excluding the 36 N-terminal amino acid residues ofsuch sequence, encoded by the longest open reading frame in the cDNAinsert in the vector deposited as ATCC Deposit No. 203587, (3) theentire amino acid sequence, or the entire amino acid sequence excludingthe N-terminal amino acid residue of such sequence, encoded by thelongest open reading frame in the cDNA insert in the vector deposited asATCC Deposit No. 203589, (4) the entire amino acid sequence, or theentire amino acid sequence excluding the N-terminal amino acid residueof such sequence, encoded by the longest open reading frame in the cDNAinsert in the vector deposited as ATCC Deposit No. 203590, (5) theentire amino acid sequence, or the entire amino acid sequence excludingthe N-terminal amino acid residue of such sequence, or the entire aminoacid sequence excluding the 34 N-terminal amino acid residues of suchsequence, encoded by the longest open reading frame in the cDNA insertin the vector deposited as ATCC Deposit No. 203846, and (6) the entireamino acid sequence, or the entire amino acid sequence excluding theN-terminal amino acid residue of such sequence, or the entire amino acidsequence excluding the 45 N-terminal amino acid residues of suchsequence, encoded by the longest open reading frame in the cDNA insertin the vector deposited as ATCC Deposit No. 203973.

In a preferred embodiment, the IL-1lp polypeptide comprises an aminoacid sequence selected from the group consisting of: (1) the entireamino acid sequence encoded by the longest open reading frame in thecDNA insert in the vector deposited as ATCC Deposit No. 203588, (2) theentire amino acid sequence, or the entire amino acid sequence excludingthe 36 N-terminal amino acid residues of such sequence, encoded by thelongest open reading frame in the cDNA insert in the vector deposited asATCC Deposit No. 203587, (3) the entire amino acid sequence, or theentire amino acid sequence excluding the N-terminal amino acid residueof such sequence, encoded by the longest open reading frame in the cDNAinsert in the vector deposited as ATCC Deposit No. 203589, (4) theentire amino acid sequence, or the entire amino acid sequence excludingthe N-terminal amino acid residue of such sequence, encoded by thelongest open reading frame in the cDNA insert in the vector deposited asATCC Deposit No. 203590, (5) the entire amino acid sequence, or theentire amino acid sequence excluding the N-terminal amino acid residueof such sequence, or the entire amino acid sequence excluding the 34N-terminal amino acid residues of such sequence, encoded by the longestopen reading frame in the cDNA insert in the vector deposited as ATCCDeposit No. 203846, and (6) the entire amino acid sequence, or theentire amino acid sequence excluding the N-terminal amino acid residueof such sequence, or the entire amino acid sequence excluding the 45N-terminal amino acid residues of such sequence, encoded by the longestopen reading frame in the cDNA insert in the vector deposited as ATCCDeposit No. 203973.

In another aspect, the invention concerns an isolated IL-1lp polypeptidethat retains at least one biologic activity of a native sequence IL-1lp,such as the IL-1R binding activity of a native sequence hIL-1Ra3 ormIL-1Ra3, or the IL-18R binding activity of a native sequence hIL-1Ra1,hIL-1Ra1L, or hIL-1Ra1V, and that consists of an amino acid sequencehaving at least at or about 80% positives, or at least at or about 85%positives, or at least at or about 90% positives, or at least at orabout 95% positives to the sequence of amino acid residues from at orabout 37 to at or about 203, inclusive of FIG. 2 (SEQ ID NO:5), thesequence of amino acid residues from at or about 15 to at or about 193,inclusive of FIG. 3 (SEQ ID NO:7), the sequence of amino acid residuesfrom at or about 34 to at or about 155, inclusive of FIG. 7 (SEQ IDNO:13), the sequence of amino acid residues from at or about 34 to at orabout 155, inclusive of FIG. 9 (SEQ ID NO:16), or the sequence of aminoacid residues from at or about 46 to at or about 218, inclusive of FIG.19 (SEQ ID NO:25).

In another aspect, the invention concerns an isolated IL-1lp polypeptidethat retains at least one biologic activity of a native sequence IL-1lp,such as the IL-18R binding activity of a native sequence hIL-1Ra1,hIL-1Ra1L, or hIL-1Ra1V, and that consists of an amino acid sequencehaving at least at or about 80% positives, or at least at or about 85%positives, or at least at or about 90% positives, or at least at orabout 95% positives to the sequence of amino acid residues from at orabout 1 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19), or thesequence of amino acid residues from at or about 1 to at or about 218,inclusive of FIG. 19 (SEQ ID NO:25).

In another aspect, the invention provides an isolated IL-1lp selectedfrom the group consisting of: (1) a polypeptide, such as an hIL-1Ra1polypeptide, consisting of an amino acid sequence having a % positivesvalue of at least at or about 80%, or at least at or about 85%, or atleast at or about 90%, or at least at or about 95%, to the sequence ofamino acid residues from at or about 37 to at or about 63, inclusive ofFIG. 2 (SEQ ID NO:5); (2) a polypeptide, such as an hIL-1Ra1polypeptide, consisting of an amino acid sequence having a % positivesvalue of at least at or about 80%, or at least at or about 85%, or atleast at or about 90%, or at least at or about 95%, to the sequence ofamino acid residues from at or about 15 to at or about 53, inclusive ofFIG. 3 (SEQ ID NO:7); (3) a polypeptide, such as an hIL-1Ra2polypeptide, comprising the amino acid sequence of amino acid residuesfrom at or about 1 to at or about 134, inclusive of FIG. 5 (SEQ IDNO:10); (4) a polypeptide comprising the amino acid sequence of aminoacid residues from at or about 10 to at or about 134, inclusive of FIG.5 (SEQ ID NO:10); (5) a polypeptide, such as an hIL-1Ra2 polypeptide,consisting of the amino acid sequence of amino acid residues from at orabout 27 to at or about 134, inclusive of FIG. 5 (SEQ ID NO:10); (6) apolypeptide, such as an hIL-1Ra2 fusion variant polypeptide, consistingof a native amino acid sequence of hIL-1Ra2 consisting of amino acidresidues from at or about 27 to at or about 134, inclusive of FIG. 5(SEQ ID NO:10) fused at its N-terminus or C-terminus to a heterologousamino acid or amino acid sequence; (7) a polypeptide, such as anhIL-1Ra3 polypeptide, consisting of an amino acid sequence having a %positives value of at least at or about 80%, or at least at or about85%, or at least at or about 90%, or at least at or about 95%, to thesequence of amino acid residues from at or about 95 to at or about 134,inclusive of FIG. 7 (SEQ ID NO:13); and (8) a polypeptide, such as amIL-1Ra3 polypeptide, consisting of an amino acid sequence having a %positives value of at least at or about 80%, or at least at or about85%, or at least at or about 90%, or at least at or about 95%, to thesequence of amino acid residues from at or about 95 to at or about 134,inclusive of FIG. 9 (SEQ ID NO:16).

In another aspect, the invention provides an isolated IL-1lp selectedfrom the group consisting of: (1) a polypeptide, such as an hIL-1Ra1Lpolypeptide, consisting of an amino acid sequence having a % positivesvalue of at least at or about 80%, or at least at or about 85%, or atleast at or about 90%, or at least at or about 95%, to the sequence ofamino acid residues from at or about 27 to at or about 44, inclusive ofFIG. 15 (SEQ ID NO:19); (2) a polypeptide, such as an hIL-1Ra1S fusionvariant polypeptide, consisting of a native amino acid sequence ofhIL-1Ra1S consisting of amino acid residues from at or about 39 to at orabout 167, inclusive of FIG. 16 (SEQ ID NO:21) fused at its N-terminusor C-terminus to a heterologous amino acid or amino acid sequence; (3) apolypeptide, such as an hIL-1Ra1S fusion variant polypeptide, consistingof a native amino acid sequence of hIL-1Ra1S consisting of amino acidresidues from at or about 47 to at or about 167, inclusive of FIG. 16(SEQ ID NO:21) fused at its N-terminus or C-terminus to a heterologousamino acid or amino acid sequence; (4) a polypeptide, such as anhIL-1Ra1S polypeptide, consisting of amino acid residues from at orabout 39 to at or about 167, inclusive of FIG. 16 (SEQ ID NO:21); (5) apolypeptide, such as an hIL-1Ra1S polypeptide, consisting of amino acidresidues from at or about 47 to at or about 167, inclusive of FIG. 16(SEQ ID NO:21); and (6) a polypeptide, such as an hIL-1Ra1V polypeptide,consisting of an amino acid sequence having a % positives value of atleast at or about 80%, or at least at or about 85%, or at least at orabout 90%, or at least at or about 95%, to the sequence of amino acidresidues from at or about 37 to at or about 55, inclusive of FIG. 19(SEQ ID NO:25).

In another aspect, the invention concerns an isolated IL-1lp polypeptidethat consists of an amino acid sequence having at least at or about 80%positives, or at least at or about 85% positives, or at least at orabout 90% positives, or at least at or about 95% positives to thesequence of amino acid residues from at or about 37 to at or about 203,inclusive of FIG. 2 (SEQ ID NO:5), the sequence of amino acid residuesfrom at or about 15 to at or about 193, inclusive of FIG. 3 (SEQ IDNO:7), the sequence of amino acid residues from at or about 34 to at orabout 155, inclusive of FIG. 7 (SEQ ID NO:13), the sequence of aminoacid residues from at or about 34 to at or about 155, inclusive of FIG.9 (SEQ ID NO:16), or the sequence of amino acid residues from at orabout 46 to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25).

In another aspect, the invention concerns an isolated IL-1lp polypeptidethat consists of an amino acid sequence having at least at or about 80%positives, or at least at or about 85% positives, or at least at orabout 90% positives, or at least at or about 95% positives to thesequence of amino acid residues from at or about 1 to at or about 207,inclusive of FIG. 15 (SEQ ID NO:19), or the sequence of amino acidresidues from at or about 1 to at or about 218, inclusive of FIG. 19(SEQ ID NO:25).

In a further aspect, the invention concerns an isolated IL-1lppolypeptide that retains at least one biologic activity of a nativesequence IL-1lp, such as the IL-1R binding activity of a native sequencehIL-1Ra3 or mIL-1Ra3, or the IL-18R binding activity of a nativesequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, and that consists of anamino acid sequence that has at least at or about 80% positives, or atleast at or about 85% positives, or at least at or about 90% positives,or at least at or about 95% positives to the amino acid sequence of anIL-1lp, such as a mature IL-1lp polypeptide, encoded by the cDNA insertin the vector deposited as ATCC Deposit No. 203588 (DNA85066-2534), ATCCDeposit No. 203587 (DNA96786-2534), ATCC Deposit No. 203589(DNA96787-2534), ATCC Deposit No. 203590 (DNA92505-2534), ATCC DepositNo. 203846 (DNA102043-2534), or ATCC Deposit No. 203973(DNA114876-2534).

In a further aspect, the invention concerns an isolated IL-1lppolypeptide that retains at least one biologic activity of a nativesequence IL-1lp, such as the IL-1R binding activity of a native sequencehIL-1Ra3 or mIL-1Ra3, or the IL-18R binding activity of a nativesequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, and that consists of anamino acid sequence that has at least at or about 80% positives, or atleast at or about 85% positives, or at least at or about 90% positives,or at least at or about 95% positives to the entire amino acid sequenceencoded by the longest open reading frame in the cDNA insert of a vectorselected from the group consisting of the vectors deposited as ATCCDeposit No. 203588 (DNA85066-2534), ATCC Deposit No. 203587(DNA96786-2534), ATCC Deposit No. 203589 (DNA96787-2534), ATCC DepositNo. 203590 (DNA92505-2534), ATCC Deposit No. 203846 (DNA102043-2534),and ATCC Deposit No. 203973 (DNA114876-2534).

In another aspect, the invention concerns an isolated IL-1lp polypeptidethat retains at least one biologic activity of a native sequence IL-1lp,such as the IL-1R binding activity of a native sequence IL-1Ra3 ormIL-1Ra3, or the IL-18R binding activity of a native sequence hIL-1Ra1,hIL-1Ra1L, or hIL-1Ra1V, and that consists of an amino acid sequencehaving at least at or about 80% positives, or at least at or about 85%positives, or at least at or about 90% positives, or at least at orabout 95% positives to an amino acid sequence selected from the groupconsisting of: (1) the entire amino acid sequence encoded by the longestopen reading frame in the cDNA insert in the vector deposited as ATCCDeposit No. 203588, (2) the entire amino acid sequence, or the entireamino acid sequence excluding the 36 N-terminal amino acid residues ofsuch sequence, encoded by the longest open reading frame in the cDNAinsert in the vector deposited as ATCC Deposit No. 203587, (3) theentire amino acid sequence, or the entire amino acid sequence excludingthe N-terminal amino acid residue of such sequence, encoded by thelongest open reading frame in the cDNA insert in the vector deposited asATCC Deposit No. 203589, (4) the entire amino acid sequence, or theentire amino acid sequence excluding the N-terminal amino acid residueof such sequence, encoded by the longest open reading frame in the cDNAinsert in the vector deposited as ATCC Deposit No. 203590, (5) theentire amino acid sequence, or the entire amino acid sequence excludingthe N-terminal amino acid residue of such sequence, or the entire aminoacid sequence excluding the 34 N-terminal amino acid residues of suchsequence, encoded by the longest open reading frame in the cDNA insertin the vector deposited as ATCC Deposit No. 203846, and (6) the entireamino acid sequence, or the entire amino acid sequence excluding theN-terminal amino acid residue of such sequence, or the entire amino acidsequence excluding the 45 N-terminal amino acid residues of suchsequence, encoded by the longest open reading frame in the cDNA insertin the vector deposited as ATCC Deposit No. 203973.

In a further aspect, the invention concerns an isolated IL-1lppolypeptide that consists of an amino acid sequence that has at least ator about 80% positives, or at least at or about 85% positives, or atleast at or about 90% positives, or at least at or about 95% positivesto the amino acid sequence of an IL-1lp, such as a mature IL-1lppolypeptide, encoded by the cDNA insert in the vector deposited as ATCCDeposit No. 203588 (DNA85066-2534), ATCC Deposit No. 203587(DNA96786-2534), ATCC Deposit No. 203589 (DNA96787-2534), ATCC DepositNo. 203590 (DNA92505-2534), ATCC Deposit No. 203846 (DNA102043-2534), orATCC Deposit No. 203973 (DNA114876-2534).

In a further aspect, the invention concerns an isolated IL-1lppolypeptide that consists of an amino acid sequence that has at least ator about 80% positives, or at least at or about 85% positives, or atleast at or about 90% positives, or at least at or about 95% positivesto the entire amino acid sequence encoded by the longest open readingframe in the cDNA insert of a vector selected from the group consistingof the vectors deposited as ATCC Deposit No. 203588 (DNA85066-2534),ATCC Deposit No. 203587 (DNA96786-2534), ATCC Deposit No. 203589(DNA96787-2534), ATCC Deposit No. 203590 (DNA92505-2534), ATCC DepositNo. 203846 (DNA102043-2534), and ATCC Deposit No. 203973(DNA114876-2534).

In another aspect, the invention concerns an isolated IL-1lp polypeptidethat consists of an amino acid sequence having at least at or about 80%positives, or at least at or about 85% positives, or at least at orabout 90% positives, or at least at or about 95% positives to an aminoacid sequence selected from the group consisting of: (1) the entireamino acid sequence encoded by the longest open reading frame in thecDNA insert in the vector deposited as ATCC Deposit No. 203588, (2) theentire amino acid sequence, or the entire amino acid sequence excludingthe 36 N-terminal amino acid residues of such sequence, encoded by thelongest open reading frame in the cDNA insert in the vector deposited asATCC Deposit No. 203587, (3) the entire amino acid sequence, or theentire amino acid sequence excluding the N-terminal amino acid residueof such sequence, encoded by the longest open reading frame in the cDNAinsert in the vector deposited as ATCC Deposit No. 203589, (4) theentire amino acid sequence, or the entire amino acid sequence excludingthe N-terminal amino acid residue of such sequence, encoded by thelongest open reading frame in the cDNA insert in the vector deposited asATCC Deposit No. 203590, (5) the entire amino acid sequence, or theentire amino acid sequence excluding the N-terminal amino acid residueof such sequence, or the entire amino acid sequence excluding the 34N-terminal amino acid residues of such sequence, encoded by the longestopen reading frame in the cDNA insert in the vector deposited as ATCCDeposit No. 203846, and (6) the entire amino acid sequence, or theentire amino acid sequence excluding the N-terminal amino acid residueof such sequence, or the entire amino acid sequence excluding the 45N-terminal amino acid residues of such sequence, encoded by the longestopen reading frame in the cDNA insert in the vector deposited as ATCCDeposit No. 203973.

In another aspect, the invention provides an isolated polypeptide, suchas an hIL-1Ra3 polypeptide, consisting of an amino acid sequence havinga % positives value of at least at or about 80%, or at least at or about85%, or at least at or about 90%, or at least at or about 95%, to thesequence of amino acid residues from at or about 80 to at or about 155,inclusive of FIG. 7 (SEQ ID NO:13).

In yet another aspect, the invention concerns an isolated IL-1lppolypeptide comprising an amino acid sequence selected from the groupconsisting of: (1) amino acid residues from at or about 37 to at orabout 63, inclusive of FIG. 2 (SEQ ID NO:5); (2) amino acid residuesfrom at or about 15 to at or about 53, inclusive of FIG. 3 (SEQ IDNO:7); (3) amino acid residues from at or about 80 to at or about 155,inclusive of FIG. 7 (SEQ ID NO:13); and (4) amino acid residues from ator about 95 to at or about 155, inclusive of FIG. 9 (SEQ ID NO:16), or afragment of such IL-1lp polypeptide that coincides with a stretch of atleast about 10 contiguous amino acids in such amino acid sequence,wherein the IL-1lp polypeptide or fragment thereof is sufficient toprovide a binding site for an anti-IL-1lp antibody. Preferably, theIL-1lp fragment retains at least one biologic activity of a nativesequence IL-1lp polypeptide.

In yet another aspect, the invention concerns an isolated IL-1lppolypeptide comprising an amino acid sequence selected from the groupconsisting of: (1) amino acid residues from at or about 26 to at orabout 44, inclusive of FIG. 15 (SEQ ID NO:19), (2) amino acid residuesfrom at or about 26 to at or about 78, inclusive of FIG. 19 (SEQ IDNO:25), and (3) amino acid residues from at or about 46 to at or about89, inclusive of FIG. 19 (SEQ ID NO:25), or a fragment of such IL-1lppolypeptide that coincides with a stretch of at least about 10contiguous amino acids in such amino acid sequence, wherein the IL-1lppolypeptide or fragment thereof is sufficient to provide a binding sitefor an anti-IL-1lp antibody. Preferably, the IL-1lp fragment retains atleast one biologic activity of a native sequence IL-1lp polypeptide.

In a further aspect, the invention provides an isolated IL-1lppolypeptide selected from the group consisting of: (1) a polypeptide,such as an hIL-1Ra1 polypeptide, comprising amino acid residues from ator about 37 to at or about 63, inclusive of FIG. 2 (SEQ ID NO:5); (2) apolypeptide, such as an hIL-1Ra1 polypeptide, comprising amino acidresidues from at or about 15 to at or about 53, inclusive of FIG. 3 (SEQID NO:7); (3) a polypeptide, such as an hIL-1Ra3 polypeptide, comprisingamino acid residues from at or about 95 to at or about 134, inclusive ofFIG. 7 (SEQ ID NO:13); and (4) a polypeptide, such as a mIL-1Ra3polypeptide, comprising amino acid residues from at or about 95 to at orabout 134, inclusive of FIG. 9 (SEQ ID NO:16).

In a further aspect, the invention provides an isolated IL-1lppolypeptide selected from the group consisting of: (1) a polypeptide,such as an hIL-1Ra1 polypeptide, comprising amino acid residues from ator about 37 to at or about 203, inclusive of FIG. 2 (SEQ ID NO:5); (2) apolypeptide, such as an hIL-1Ra1 polypeptide, comprising amino acidresidues from at or about 15 to at or about 193, inclusive of FIG. 3(SEQ ID NO:7); (3) a polypeptide, such as an hIL-1Ra3 polypeptide,comprising amino acid residues from at or about 34 to at or about 155,inclusive of FIG. 7 (SEQ ID NO:13); and (3) a polypeptide, such as amIL-1Ra3 polypeptide, comprising amino acid residues from at or about 34to at or about 155, inclusive of FIG. 9 (SEQ ID NO:16).

In a further aspect, the invention provides an isolated polypeptide,such as an hIL-1Ra3 polypeptide, comprising amino acid residues from ator about 80 to at or about 155, inclusive of FIG. 7 (SEQ ID NO:13).

In a further aspect, the invention provides an isolated IL-1lppolypeptide selected from the group consisting of: (1) a polypeptide,such as an hIL-1Ra1L polypeptide, comprising amino acid residues from ator about 26 to at or about 44, inclusive of FIG. 15 (SEQ ID NO:19); (2)a polypeptide, such as an hIL-1Ra1L polypeptide, comprising amino acidresidues from at or about 1 to at or about 44, inclusive of FIG. 15 (SEQID NO:19); (3) a polypeptide, such as an hIL-1Ra1S polypeptide,comprising amino acid residues from at or about 1 to at or about 38,inclusive of FIG. 16 (SEQ ID NO:21); (4) a polypeptide, such as anhIL-1Ra1V polypeptide, comprising amino acid residues from at or about37 to at or about 55, inclusive of FIG. 19 (SEQ ID NO:25); (5) apolypeptide, such as an hIL-1Ra1V polypeptide, comprising amino acidresidues from at or about 12 to at or about 55, inclusive of FIG. 19(SEQ ID NO:25); and (6) a polypeptide, such as an hIL-1Ra1V polypeptide,comprising amino acid residues from at or about 1 to at or about 55,inclusive of FIG. 19 (SEQ ID NO:25).

In a further aspect, the invention provides an isolated IL-1lppolypeptide selected from the group consisting of: (1) a polypeptide,such as an hIL-1Ra1L fusion variant polypeptide, consisting of a nativeamino acid sequence of hIL-1Ra1L consisting of amino acid residues fromat or about 1 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19)fused at its N-terminus or C-terminus to a heterologous amino acid oramino acid sequence; (2) a polypeptide, such as an hIL-1Ra1L fusionvariant polypeptide, consisting of a native amino acid sequence ofhIL-1Ra1L consisting of amino acid residues from at or about 26 to at orabout 207, inclusive of FIG. 15 (SEQ ID NO:19) fused at its N-terminusor C-terminus to a heterologous amino acid or amino acid sequence; (3) apolypeptide, such as an hIL-1Ra1L polypeptide, consisting of amino acidresidues from at or about 1 to at or about 207, inclusive of FIG. 15(SEQ ID NO:19); (4) a polypeptide, such as an hIL-1Ra1L polypeptide,consisting of amino acid residues from at or about 26 to at or about207, inclusive of FIG. 15 (SEQ ID NO:19); (5) a polypeptide, such as anhIL-1Ra1V fusion variant polypeptide, consisting of a native amino acidsequence of hIL-1Ra1V consisting of amino acid residues from at or about1 to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25) fused at itsN-terminus or C-terminus to a heterologous amino acid or amino acidsequence; (6) a polypeptide, such as an hIL-1Ra1V fusion variantpolypeptide, consisting of a native amino acid sequence of hIL-1Ra1Vconsisting of amino acid residues from at or about 12 to at or about218, inclusive of FIG. 19 (SEQ ID NO:25) fused at its N-terminus orC-terminus to a heterologous amino acid or amino acid sequence; (7) apolypeptide, such as an hIL-1Ra1V fusion variant polypeptide, consistingof a native amino acid sequence of hIL-1Ra1V consisting of amino acidresidues from at or about 37 to at or about 218, inclusive of FIG. 19(SEQ ID NO:25) fused at its N-terminus or C-terminus to a heterologousamino acid or amino acid sequence; (8) a polypeptide, such as anhIL-1Ra1V fusion variant polypeptide, consisting of a native amino acidsequence of hIL-1Ra1V consisting of amino acid residues from at or about46 to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25) fused at itsN-terminus or C-terminus to a heterologous amino acid or amino acidsequence; (9) a polypeptide, such as an hIL-1Ra1V polypeptide,consisting of the amino acid sequence of amino acid residues from at orabout 1 to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25); (10) apolypeptide, such as an hIL-1Ra1V polypeptide, consisting of the aminoacid sequence of amino acid residues from at or about 12 to at or about218, inclusive of FIG. 19 (SEQ ID NO:25); (11) a polypeptide, such as anhIL-1Ra1V polypeptide, consisting of the amino acid sequence of aminoacid residues from at or about 37 to at or about 218, inclusive of FIG.19 (SEQ ID NO:25); and (12) a polypeptide, such as an hIL-1Ra1Vpolypeptide, consisting of the amino acid sequence of amino acidresidues from at or about 46 to at or about 218, inclusive of FIG. 19(SEQ ID NO:25).

In a further aspect, the invention provides an isolated IL-1lppolypeptide selected from the group consisting of: (1) a polypeptide,such as an hIL-1Ra1L polypeptide, comprising amino acid residues from ator about 1 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19); (2)a polypeptide, such as an hIL-1Ra1L polypeptide, comprising amino acidresidues from at or about 26 to at or about 207, inclusive of FIG. 15(SEQ ID NO:19); (3) a polypeptide, such as an hIL-1Ra1S polypeptide,comprising amino acid residues from at or about 1 to at or about 167,inclusive of FIG. 16 (SEQ ID NO:21); (4) a polypeptide, such as anhIL-1Ra1S polypeptide, comprising amino acid residues from at or about26 to at or about 167, inclusive of FIG. 16 (SEQ ID NO:21); (5) apolypeptide, such as an hIL-1Ra1V polypeptide, comprising amino acidresidues from at or about 1 to at or about 218, inclusive of FIG. 19(SEQ ID NO:25); and (6) a polypeptide, such as an hIL-1Ra1V polypeptide,comprising amino acid residues from at or about 12 to at or about 218,inclusive of FIG. 19 (SEQ ID NO:25).

In a further aspect, the invention provides an isolated IL-1lppolypeptide selected from the group consisting of: (1) a polypeptide,such as an hIL-1Ra1V polypeptide, comprising amino acid residues from ator about 37 to at or about 218, inclusive of FIG. 19 (SEQ ID NO:25); and(2) a polypeptide, such as an hIL-1Ra1V polypeptide, comprising aminoacid residues from at or about 46 to at or about 218, inclusive of FIG.19 (SEQ ID NO:25).

In a further aspect, the invention provides an isolated IL-1lppolypeptide selected from the group consisting of: (1) a polypeptideconsisting of the amino acid sequence of amino acid residues 10 to 134,inclusive of FIG. 5 (SEQ ID NO:10) fused at its N-terminus or C-terminusto a heterologous amino acid or amino acid sequence; (2) a polypeptideconsisting of the amino acid sequence of amino acid residues 10 to 134,inclusive of FIG. 5 (SEQ ID NO:10); (3) a polypeptide, such as anhIL-1Ra3 polypeptide, consisting of a native amino acid sequence ofhIL-1Ra3 consisting of amino acid residues 2 to 155, inclusive of FIG. 7(SEQ ID NO:13) fused at its N-terminus or C-terminus to a heterologousamino acid or amino acid sequence; (4) a polypeptide, such as anhIL-1Ra3 polypeptide, consisting of the amino acid sequence of aminoacid residues from 2 to 155, inclusive of FIG. 7 (SEQ ID NO:13); (5) apolypeptide, such as a mIL-1Ra3 polypeptide, consisting of a nativeamino acid sequence of mIL-1Ra3 consisting of amino acid residues 2 to155, inclusive of FIG. 9 (SEQ ID NO:16) fused at its N-terminus orC-terminus to a heterologous amino acid or amino acid sequence; (6) apolypeptide, such as a mIL-1Ra3 polypeptide, consisting of the aminoacid sequence of amino acid residues 2 to 155, inclusive of FIG. 9 (SEQID NO:16).

In a further aspect, the invention provides an isolated IL-1lppolypeptide selected from the group consisting of: (1) a polypeptidecomprising the amino acid sequence of amino acid residues 10 to 134,inclusive of FIG. 5 (SEQ ID NO:10); (2) a polypeptide, such as anhIL-1Ra3 polypeptide, comprising the amino acid sequence of amino acidresidues 2 to 155, inclusive of FIG. 7 (SEQ ID NO:13); and (3) apolypeptide, such as a mIL-1Ra3 polypeptide, comprising the amino acidsequence of amino acid residues from 2 to 155, inclusive of FIG. 9 (SEQID NO:16).

In a still further aspect, the invention provides an isolated IL-1lppolypeptide that is the same as a mature polypeptide encoded by the cDNAinsert of a vector selected from the group consisting of the vectorsdeposited as ATCC Deposit Nos. 203588, 203587, 203586, 203589, and203590.

In a still further aspect, the invention provides an isolated IL-1lppolypeptide that is the same as a mature polypeptide encoded by the cDNAinsert of a vector selected from the group consisting of the vectorsdeposited as ATCC Deposit Nos. 203846, 203855 and 203973.

In a still further aspect, the invention provides an isolatedpolypeptide comprising the entire amino acid sequence encoded by thelongest open reading frame in the cDNA insert of a vector selected fromthe group consisting of the vectors deposited as ATCC Deposit Nos.203588, 203586, 203589 and 203590.

In a still further aspect, the invention provides an isolatedpolypeptide comprising the entire amino acid sequence encoded by thelongest open reading frame in the cDNA insert of a vector selected fromthe group consisting of the vectors deposited as ATCC Deposit Nos.203846, 203855 and 203973.

In another aspect, the invention provides a polypeptide that is producedby (i) hybridizing a test DNA molecule under stringent conditions with(a) a DNA molecule encoding an amino acid sequence selected from thegroup consisting of: (1) amino acid residues from at or about 37 to ator about 203, inclusive of FIG. 2 (SEQ ID NO:5), (2) amino acid residuesfrom at or about 15 to at or about 193, inclusive of FIG. 3 (SEQ IDNO:7), (3) amino acid residues from at or about 2 to at or about 155,inclusive of FIG. 7 (SEQ ID NO:13), and (4) amino acid residues from ator about 2 to at or about 155, inclusive of FIG. 9 (SEQ ID NO:16), or(b) the complement of the DNA molecule of (a), and if the test DNAmolecule encodes an IL-1lp polypeptide that retains at least onebiologic activity of a native sequence IL-1lp, such as the IL-1R bindingactivity of a native sequence hIL-1Ra3 or mIL-1Ra3, or the IL-18Rbinding activity of a native sequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V,and if the test DNA molecule has at least at or about an 80% sequenceidentity, or at least at or about an 85% sequence identity, or at leastat or about a 90% sequence identity, or at least at or about a 95%sequence identity to the DNA molecule of (a) or (b), (ii) culturing ahost cell comprising the test DNA molecule under conditions suitable forexpression of the IL-1lp polypeptide, and (iii) recovering the IL-1lppolypeptide from the cell culture.

In a still further aspect, the invention provides a polypeptide that isproduced by (i) hybridizing a test DNA molecule under stringentconditions with (a) a DNA molecule encoding an amino acid sequenceselected from the group consisting of: (1) amino acid residues from ator about 26 to at or about 207, inclusive of FIG. 15 (SEQ ID NO:19), (2)amino acid residues from at or about 1 to at or about 218, inclusive ofFIG. 19 (SEQ ID NO:25), (3) amino acid residues from at or about 12 toat or about 218, inclusive of FIG. 19 (SEQ ID NO:25), (4) amino acidresidues from at or about 37 to at or about 218, inclusive of FIG. 19(SEQ ID NO:25), and (5) amino acid residues from at or about 46 to at orabout 218, inclusive of FIG. 19 (SEQ ID NO:25), or (b) the complement ofthe DNA molecule of (a), and if the test DNA molecule encodes an IL-1lppolypeptide that retains at least one biologic activity of a nativesequence IL-1lp, such as the IL-18R binding activity of a nativesequence hIL-1Ra1, hIL-1Ra1L, or hIL-1Ra1V, and if the test DNA moleculehas at least at or about an 80% sequence identity, or at least at orabout an 85% sequence identity, or at least at or about a 90% sequenceidentity, or at least at or about a 95% sequence identity to the DNAmolecule of (a) or (b), (ii) culturing a host cell comprising the testDNA molecule under conditions suitable for expression of the IL-1lppolypeptide, and (iii) recovering the IL-1lp polypeptide from the cellculture.

A. Preparation of IL-1lp

The description below relates primarily to production of IL-1lp byculturing cells transformed or transfected with a vector containingIL-1lp nucleic acid. It is, of course, contemplated that alternativemethods, which are well known in the art, may be employed to prepareIL-1lp. For instance, the IL-1lp sequence, or portions thereof, may beproduced by direct peptide synthesis using solid-phase techniques [see,e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co.,San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc.,85:2149-2154 (1963)]. In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may beaccomplished, for instance, using an Applied Biosystems PeptideSynthesizer (Foster City, Calif.) using manufacturer's instructions.Various portions of the IL-1lp may be chemically synthesized separatelyand combined using chemical or enzymatic methods to produce thefull-length IL-1lp.

1. Isolation of DNA Encoding IL-1lp

DNA encoding IL-1lp may be obtained from a cDNA library prepared fromtissue believed to possess the IL-1lp mRNA and to express it at adetectable level. Accordingly, human IL-1lp DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The IL-1lp-encoding gene may also be obtainedfrom a genomic library or by oligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to the IL-1lpor oligonucleotides of at least about 20-80 bases) designed to identifythe gene of interest or the protein encoded by it. Screening the cDNA orgenomic library with the selected probe may be conducted using standardprocedures, such as described in Sambrook et al., Molecular Cloning: ALaboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).An alternative means to isolate the gene encoding IL-1lp is to use PCRmethodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined through sequence alignment using computer software programssuch as BLAST, BLAST2, ALIGN-2, DNAstar, and INHERIT which employvarious algorithms to measure homology.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for IL-1lp production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Methods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Depending on the host cell used,transformation is performed using standard techniques appropriate tosuch cells. The calcium treatment employing calcium chloride, asdescribed in Sambrook et al., supra, or electroporation is generallyused for prokaryotes or other cells that contain substantial cell-wallbarriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635).

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forIL-1lp-encoding vectors. Saccharomyces cerevisiae is a commonly usedlower eukaryotic host microorganism.

Suitable host cells for the expression of glycosylated IL-1lp arederived from multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9, as wellas plant cells. Examples of useful mammalian host cell lines includeChinese hamster ovary (CHO) and COS cells. More specific examplesinclude monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinese hamster ovary cells/-DHFR(CHO, Urlaub and Chasin, Proc.Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather,Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75);human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding IL-1lp may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The IL-1lp may be produced recombinantly not only directly, but also asa fusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe IL-1lp-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, 1 pp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), orthe signal described in WO 90/13646 published 15 Nov. 1990. In mammaliancell expression, mammalian signal sequences may be used to directsecretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up theIL-1lp-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10: 157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the IL-1lp-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encodingIL-1lp.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

IL-1lp transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the IL-1lp by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theIL-1lp coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding IL-1lp.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of IL-1lp in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceIL-1lp polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to IL-1lpDNA and encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of IL-1lp may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of IL-1lp can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents. It may bedesired to purify IL-1lp from recombinant cell proteins or polypeptides.The following procedures are exemplary of suitable purificationprocedures: by fractionation on an ion-exchange column; ethanolprecipitation; reverse phase HPLC; chromatography on silica or on acation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; gel filtration using, for example, Sephadex G-75;protein A Sepharose columns to remove contaminants such as IgG; andmetal chelating columns to bind epitope-tagged forms of the IL-1lp.Various methods of protein purification may be employed and such methodsare known in the art and described for example in Deutscher, Methods inEnzymology, 182 (1990); Scopes, Protein Purification: Principles andPractice, Springer-Verlag, New York (1982). The purification step(s)selected will depend, for example, on the nature of the productionprocess used and the particular IL-1lp produced.

B. Activity Assays for IL-1lp Variants

The biological activity or activities of a particular IL-1lp variantpolypeptide can be characterized using a variety of in vitro assaysknown in the art. For example, the ability of an hIL-1Ra3 variantpolypeptide or a mIL-1Ra3 variant polypeptide to bind IL-1R can beassayed using a radioimmunoprecipitation assay wherein IL-1Rextracellular domain (ECD) fused to the Fc region of humanimmunoglobulin G (IL-1R ECD-Fc) (which can be prepared, e.g., asdescribed in Examples 9 and 10 below) is incubated in solution withradiolabeled hIL-1Ra3 variant polypeptide or mIL-1Ra3 variantpolypeptide to form labeled complexes, followed by immunoprecipitationof the labeled complexes with goat anti-human IgG Fc and quantitation ofradioactivity in the precipitate. In another example, an hIL-1Ra3variant polypeptide-FLAG tag fusion protein-encoding DNA and an IL-1RECD-Fc encoding DNA can be coexpressed in a host cell and secreted intothe cell's culture medium, followed by immunoprecipitation of culturesupernatant with protein G-sepharose and identification of boundhIL-1Ra3 variant polypeptide-FLAG tag fusion protein by immunoblottingwith anti-FLAG monoclonal antibody, essentially as described in Example9 below.

In another embodiment, the ability of an hIL-1Ra3 variant polypeptide ora mIL-1Ra3 variant polypeptide to inhibit the binding of IL-1 to IL-1Rcan be assayed using a competitive binding assay. For example, aradioimmunoprecipitation assay can be employed wherein IL-1R ECD-Fc isincubated in solution of radiolabeled IL-1 with or without unlabeledhIL-1Ra3 variant polypeptide or unlabeled mIL-1Ra3 variant polypeptideto form labeled or unlabeled complexes, followed by immunoprecipitationof complexes with anti-human IgG Fc and quantitation of radioactivity inthe precipitate. If the presence of unlabeled hIL-1Ra3 variantpolypeptide or unlabeled mIL-1Ra3 variant polypeptide in the incubationsolution diminishes the radioactivity measured in the resultingimmunoprecipitate, the hIL-1Ra3 variant polypeptide or mIL-1Ra3 variantpolypeptide in question qualifies as an inhibitor of IL-1 binding toIL-1R. In yet another embodiment, IL-1R ECD-Fc and an hIL-1Ra3variant-FLAG tag fusion protein or mIL-1Ra3 variant-FLAG tag fusionprotein are obtained by recombinant expression in separate cell cultures(essentially as described in Example 10 below), IL-1 and IL-1R ECD-Fcare admixed together with or without the hIL-1Ra3 variant-FLAG tagfusion protein or mIL-1Ra3 variant-FLAG tag fusion protein and incubatedin solution, the incubation solution is immunoprecipitated with proteinG-sepharose, and the bound hIL-1Ra3 variant-FLAG tag fusion protein ormIL-1Ra3 variant-FLAG tag fusion protein is identified by immunoblottingwith anti-FLAG monoclonal antibody. If the presence of IL-1 in theincubation solution diminishes the signal detected by anti-FLAGimmunoblotting, the hIL-1Ra3 variant polypeptide or mIL-1Ra3 variantpolypeptide in question qualifies as an inhibitor of IL-1 binding toIL-1R.

Similarly, the biological activity or activities of a particularhIL-1Ra1 variant polypeptide can be determined by using a variety of invitro assays known in the art. For example, the ability of an hIL-1Ra1variant polypeptide to bind IL-18R can be assayed using aradioimmunoprecipitation assay wherein IL-18R extracellular domain (ECD)fused to the Fc region of human immunoglobulin G (IL-18R ECD-Fc) (whichcan be prepared, e.g., as described in Examples 9 and 10 below) isincubated in solution with radiolabeled hIL-1Ra1 variant polypeptide toform labeled complex, followed by immunoprecipitation of the labeledcomplex with goat anti-human IgG Fc and quantitation of radioactivity inthe precipitate. In another example, an hIL-1Ra1 variantpolypeptide-FLAG tag fusion protein-encoding DNA and an IL-18R ECD-Fcencoding DNA can be coexpressed in a host cell and secreted into thecell's culture medium, followed by immunoprecipitation of culturesupernatant with protein G-sepharose and identification of boundhIL-1Ra1 variant polypeptide-FLAG tag fusion protein by immunoblottingwith anti-FLAG monoclonal antibody, essentially as described in Example9 below.

In another embodiment, the ability of an hIL-1Ra1 variant polypeptide toinhibit the binding of IL-18 to IL-18R can be assayed using acompetitive binding assay. For example, a radioimmunoprecipitation assaycan be employed wherein IL-18R ECD-Fc is incubated in solution ofradiolabeled IL-18 with or without unlabeled hIL-1Ra1 variantpolypeptide to form labeled or unlabeled complexes, followed byimmunoprecipitation of complexes with anti-human IgG Fc and quantitationof radioactivity in the precipitate. If the presence of unlabeledhIL-1Ra1 variant polypeptide in the incubation solution diminishes theradioactivity measured in the resulting immunoprecipitate, the hIL-1Ra1variant polypeptide in question qualifies as an inhibitor of IL-18binding to IL-18R. In yet another embodiment, IL-18R ECD-Fc and anhIL-1Ra1 variant-FLAG tag fusion protein are obtained by recombinantexpression in separate cell cultures (essentially as described inExample 10 below), IL-18 and IL-18R ECD-Fc are admixed together with orwithout the hIL-1Ra1 variant-FLAG tag fusion protein and incubated insolution, the incubation solution is immunoprecipitated with proteinG-sepharose, and the bound hIL-1Ra1 variant-FLAG tag fusion protein isidentified by immunoblotting with anti-FLAG monoclonal antibody. If thepresence of IL-18 in the incubation solution diminishes the signaldetected by anti-FLAG immunoblotting, the hIL-1Ra1 variant polypeptidein question qualifies as an inhibitor of IL-18 binding to IL-18R.

C. Uses for IL-1lp

Nucleotide sequences (or their complement) encoding IL-1lp have variousapplications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. IL-1lp nucleic acid will also beuseful for the preparation of IL-1lp polypeptides by the recombinanttechniques described herein.

The full-length native sequence IL-1lp genes of FIG. 1 (SEQ ID NO:1),FIG. 2 (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:9), FIG. 7(SEQ ID NO:12), FIG. 9 (SEQ ID NO:15), FIG. 15 (SEQ ID NO:18), and FIG.16 (SEQ ID NO:20), and FIG. 19 (SEQ ID NO:24), or portions thereof, maybe used as hybridization probes for a cDNA library to isolate thefull-length IL-1lp gene or to isolate still other genes (for instance,those encoding naturally-occurring variants of IL-1lp or IL-1lp fromother species) which have a desired sequence identity to the IL-1lpsequence disclosed in FIG. 1 (SEQ ID NO:1), FIG. 2 (SEQ ID NO:4), FIG. 3(SEQ ID NO:6), FIG. 5 (SEQ ID NO:9), FIG. 7 (SEQ ID NO:12), FIG. 9 (SEQID NO:15), FIG. 15 (SEQ ID NO:18), FIG. 16 (SEQ ID NO:20), or FIG. 19(SEQ ID NO:24). Optionally, the length of the probes will be about 20 toabout 50 bases. The hybridization probes may be derived from thenucleotide sequence of FIG. 1 (SEQ ID NO:1), FIG. 2 (SEQ ID NO:4), FIG.3 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:9), FIG. 7 (SEQ ID NO:12), FIG. 9(SEQ ID NO:15), FIG. 15 (SEQ ID NO:18), FIG. 16 (SEQ ID NO:20) or FIG.19 (SEQ ID NO:24), or from genomic sequences including promoters,enhancer elements and introns of native sequence IL-1lp. By way ofexample, a screening method will comprise isolating the coding region ofthe IL-1lp gene using the known DNA sequence to synthesize a selectedprobe of about 40 bases. Hybridization probes may be labeled by avariety of labels, including radionucleotides such as ³²P or ³⁵S, orenzymatic labels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the IL-1lp gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related IL-1lp coding sequences.

Nucleotide sequences encoding an IL-1lp can also be used to constructhybridization probes for mapping the gene which encodes that IL-1lp andfor the genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

When the coding sequences for IL-1lp encode a protein which binds toanother protein (example, where the IL-1lp binds to an IL-1 receptor orIL-18 receptor), the IL-1lp can be used in assays to identify the otherproteins or molecules involved in the binding interaction. By suchmethods, inhibitors of the receptor/ligand binding interaction can beidentified. Proteins involved in such binding interactions can also beused to screen for peptide or small molecule inhibitors or agonists ofthe binding interaction. Screening assays can be designed to find leadcompounds that mimic the biological activity of a native IL-1lp or areceptor for IL-1lp. Such screening assays will include assays amenableto high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.Small molecules contemplated include synthetic organic or inorganiccompounds. The assays can be performed in a variety of formats,including protein-protein binding assays, biochemical screening assays,immunoassays and cell based assays, which are well characterized in theart.

Nucleic acids which encode IL-1lp or its modified forms can also be usedto generate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding IL-1lp can be used to clone genomic DNAencoding IL-1lp in accordance with established techniques and thegenomic sequences used to generate transgenic animals that contain cellswhich express DNA encoding IL-1lp. Methods for generating transgenicanimals, particularly animals such as mice or rats, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009. Typically, particular cells would betargeted for IL-1lp transgene incorporation with tissue-specificenhancers. Transgenic animals that include a copy of a transgeneencoding IL-1lp introduced into the germ line of the animal at anembryonic stage can be used to examine the effect of increasedexpression of DNA encoding IL-1lp. Such animals can be used as testeranimals for reagents thought to confer protection from, for example,pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of IL-1lp can be used to constructan IL-1lp “knock out” animal which has a defective or altered geneencoding IL-1lp as a result of homologous recombination between theendogenous gene encoding IL-1lp and altered genomic DNA encoding IL-1lpintroduced into an embryonic cell of the animal. For example, cDNAencoding IL-1lp can be used to clone genomic DNA encoding IL-1lp inaccordance with established techniques. A portion of the genomic DNAencoding IL-1lp can be deleted or replaced with another gene, such as agene encoding a selectable marker which can be used to monitorintegration. Typically, several kilobases of unaltered flanking DNA(both at the 5′ and 3′ ends) are included in the vector [see e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors]. The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected[see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras [see e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the IL-1lp polypeptide.

Nucleic acid encoding the IL-1lp polypeptides may also be used in genetherapy. In gene therapy applications, genes are introduced into cellsin order to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83, 4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

The IL-1lp polypeptides of the present invention can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby the IL-1lp product hereof is combined in admixturewith a pharmaceutically acceptable carrier vehicle. Therapeuticformulations are prepared for storage by mixing the active ingredienthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrateand other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as Tween,Pluronics or PEG.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96.

An “effective amount” of the IL-1lp to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.Typically, the clinician will administer the IL-1lp until a dosage isreached that achieves the desired effect. The progress of this therapyis easily monitored by conventional assays.

In one embodiment, the invention provides a method for treating anIL-1-mediated disorder comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an IL-1lp, suchas a native sequence IL-1lp.

In another embodiment, the invention provides a method for treating anIL-1-mediated disorder comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an IL-1lpselected from the group consisting of hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V,hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.

In another embodiment, the invention provides a method for treating anIL-1-mediated disorder comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an hIL-1lp, suchas a native sequence hIL-1lp, e.g. native sequence hIL-1Ra3.

In another embodiment, the invention provides a method for treating anIL-18-mediated disorder comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an IL-1lp, suchas a native sequence IL-1lp.

In another embodiment, the invention provides a method for treating anIL-18-mediated disorder comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an IL-1lpselected from the group consisting of hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V,hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.

In another embodiment, the invention provides a method for treating anIL-18-mediated disorder comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an hIL-1lp, suchas a native sequence hIL-1lp, e.g. native sequence hIL-1Ra1.

In another embodiment, the invention provides a method for treating anIL-18-mediated disorder comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an hIL-1Ra1L,such as a native sequence hIL-1Ra1L, or an effective amount of anhIL-1Ra1V, such as a native sequence hIL-1Ra1V, or an effective amountof an hIL-1Ra1S, such as a native sequence hIL-1Ra1S.

In one embodiment, the invention provides a method for treating aninflammatory disorder comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an IL-1lp, suchas a native sequence IL-1lp.

In another embodiment, the invention provides a method for treating aninflammatory disorder comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an IL-1lpselected from the group consisting of hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V,hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.

In another embodiment, the invention provides a method for treating aninflammatory disorder comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an hIL-1lp, suchas a native sequence hIL-1lp, e.g. native sequence hIL-1Ra1 or hIL-1Ra3.

In another embodiment, the invention provides a method for treating aninflammatory disorder comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an hIL-1Ra1L,such as a native sequence hIL-1Ra1L, or an effective amount of anhIL-1Ra1V, such as a native sequence hIL-1Ra1V, or an effective amountof an hIL-1Ra1S, such as a native sequence hIL-1Ra1S.

In another embodiment, the invention provides a method for treatingasthma comprising administering to a mammal, such as human, in need ofsuch treatment an effective amount of an IL-1lp, such as a nativesequence IL-1lp.

In another embodiment, the invention provides a method for treatingasthma comprising administering to a mammal, such as human, in need ofsuch treatment an effective amount of an IL-1lp selected from the groupconsisting of hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2,hIL-1Ra3, and mIL-1Ra3.

In another embodiment, the invention provides a method for treatingasthma comprising administering to a mammal, such as human, in need ofsuch treatment an effective amount of an hIL-1lp, such as a nativesequence hIL-1lp, e.g. native sequence hIL-1Ra1 or hIL-1Ra3.

In another embodiment, the invention provides a method for treatingasthma comprising administering to a mammal, such as human, in need ofsuch treatment an effective amount of an hIL-1Ra1L, such as a nativesequence hIL-1Ra1L, or an effective amount of an hIL-1Ra1V, such as anative sequence hIL-1Ra1V, or an effective amount of an hIL-1Ra1S, suchas a native sequence hIL-1Ra1S.

In another embodiment, the invention provides a method for treatingrheumatoid arthritis comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an IL-1lp, suchas a native sequence IL-1lp.

In another embodiment, the invention provides a method for treatingrheumatoid arthritis comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an IL-1lpselected from the group consisting of hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V,hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.

In another embodiment, the invention provides a method for treatingrheumatoid arthritis comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an hIL-1lp, suchas a native sequence hIL-1lp, e.g. native sequence hIL-1Ra1 or hIL-1Ra3.

In another embodiment, the invention provides a method for treatingrheumatoid arthritis comprising administering to a mammal, such ashuman, in need of such treatment an effective amount of an hIL-1Ra1L,such as a native sequence hIL-1Ra1L, or an effective amount of anhIL-1Ra1V, such as a native sequence hIL-1Ra1V, or an effective amountof an hIL-1Ra1S, such as a native sequence hIL-1Ra1S.

In another embodiment, the invention provides a method for treatingosteoarthritis comprising administering to a mammal, such as human, inneed of such treatment an effective amount of an IL-1lp, such as anative sequence IL-1lp.

In another embodiment, the invention provides a method for treatingosteoarthritis comprising administering to a mammal, such as human, inneed of such treatment an effective amount of an IL-1lp selected fromthe group consisting of hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S,hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.

In another embodiment, the invention provides a method for treatingosteoarthritis comprising administering to a mammal, such as human, inneed of such treatment an effective amount of an hIL-1lp, such as anative sequence hIL-1lp, e.g. native sequence hIL-1Ra1 or hIL-1Ra3.

In another embodiment, the invention provides a method for treatingosteoarthritis comprising administering to a mammal, such as human, inneed of such treatment an effective amount of an hIL-1Ra1L, such as anative sequence hIL-1Ra1L, or an effective amount of an hIL-1Ra1V, suchas a native sequence hIL-1Ra1V, or an effective amount of an hIL-1Ra1S,such as a native sequence hIL-1Ra1S.

In another embodiment, the invention provides a method for treatingsepsis comprising administering to a mammal, such as human, in need ofsuch treatment an effective amount of an IL-1lp, such as a nativesequence IL-1lp.

In another embodiment, the invention provides a method for treatingsepsis comprising administering to a mammal, such as human, in need ofsuch treatment an effective amount of an IL-1lp selected from the groupconsisting of hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2,hIL-1Ra3, and mIL-1Ra3.

In another embodiment, the invention provides a method for treatingsepsis comprising administering to a mammal, such as human, in need ofsuch treatment an effective amount of an hIL-1lp, such as a nativesequence hIL-1lp, e.g. native sequence hIL-1Ra1 or hIL-1Ra3.

In another embodiment, the invention provides a method for treatingsepsis comprising administering to a mammal, such as human, in need ofsuch treatment an effective amount of an hIL-1Ra1L, such as a nativesequence hIL-1Ra1L, or an effective amount of an hIL-1Ra1V, such as anative sequence hIL-1Ra1V, or an effective amount of an hIL-1Ra1S, suchas a native sequence hIL-1Ra1S.

In another embodiment, the invention provides a method for treatingacute lung injury comprising administering to a mammal, such as human,in need of such treatment an effective amount of an IL-1lp, such as anative sequence IL-1lp.

In another embodiment, the invention provides a method for treatingacute lung injury comprising administering to a mammal, such as human,in need of such treatment an effective amount of an IL-1lp selected fromthe group consisting of hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S,hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.

In another embodiment, the invention provides a method for treatingacute lung injury comprising administering to a mammal, such as human,in need of such treatment an effective amount of an hIL-1lp, such as anative sequence hIL-1lp, e.g. native sequence hIL-1Ra1 or hIL-1Ra3.

In another embodiment, the invention provides a method for treatingacute lung injury comprising administering to a mammal, such as human,in need of such treatment an effective amount of an hIL-1Ra1L, such as anative sequence hIL-1Ra1L, or an effective amount of an hIL-1Ra1V, suchas a native sequence hIL-1Ra1V, or an effective amount of an hIL-1Ra1S,such as a native sequence hIL-1Ra1S.

In another embodiment, the invention provides a method for treatingadult respiratory distress syndrome comprising administering to amammal, such as human, in need of such treatment an effective amount ofan IL-1lp, such as a native sequence IL-1lp.

In another embodiment, the invention provides a method for treatingadult respiratory distress syndrome comprising administering to amammal, such as human, in need of such treatment an effective amount ofan IL-1lp selected from the group consisting of hIL-1Ra1, hIL-1Ra1L,hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.

In another embodiment, the invention provides a method for treatingadult respiratory distress syndrome comprising administering to amammal, such as human, in need of such treatment an effective amount ofan hIL-1lp, such as a native sequence hIL-1lp, e.g. native sequencehIL-1Ra1 or hIL-1Ra3.

In another embodiment, the invention provides a method for treatingadult respiratory distress syndrome comprising administering to amammal, such as human, in need of such treatment an effective amount ofan hIL-1Ra1L, such as a native sequence hIL-1Ra1L, or an effectiveamount of an hIL-1Ra1V, such as a native sequence hIL-1Ra1V, or aneffective amount of an hIL-1Ra1S, such as a native sequence hIL-1Ra1S.

In another embodiment, the invention provides a method for treatingidiopathic pulmonary fibrosis comprising administering to a mammal, suchas human, in need of such treatment an effective amount of an IL-1lp,such as a native sequence IL-1lp.

In another embodiment, the invention provides a method for treatingidiopathic pulmonary fibrosis comprising administering to a mammal, suchas human, in need of such treatment an effective amount of an IL-1lpselected from the group consisting of hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V,hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.

In another embodiment, the invention provides a method for treatingidiopathic pulmonary fibrosis comprising administering to a mammal, suchas human, in need of such treatment an effective amount of an hIL-1lp,such as a native sequence hIL-1lp, e.g. native sequence hIL-1Ra1 orhIL-1Ra3.

In another embodiment, the invention provides a method for treatingidiopathic pulmonary fibrosis comprising administering to a mammal, suchas human, in need of such treatment an effective amount of an hIL-1Ra1L,such as a native sequence hIL-1Ra1L, or an effective amount of anhIL-1Ra1V, such as a native sequence hIL-1Ra1V, or an effective amountof an hIL-1Ra1S, such as a native sequence hIL-1Ra1S.

In another embodiment, the invention provides a method for treating anischemic reperfusion disease, such as surgical tissue reperfusioninjury, stroke, myocardial ischemia, or acute myocardial infarction,comprising administering to a mammal, such as human, in need of suchtreatment an effective amount of an IL-1lp, such as a native sequenceIL-1lp.

In another embodiment, the invention provides a method for treating anischemic reperfusion disease, such as surgical tissue reperfusioninjury, stroke, myocardial ischemia, or acute myocardial infarction,comprising administering to a mammal, such as human, in need of suchtreatment an effective amount of an IL-1lp selected from the groupconsisting of hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2,hIL-1Ra3, and mIL-1Ra3.

In another embodiment, the invention provides a method for treating anischemic reperfusion disease, such as surgical tissue reperfusioninjury, stroke, myocardial ischemia, or acute myocardial infarction,comprising administering to a mammal, such as human, in need of suchtreatment an effective amount of an hIL-1lp, such as a native sequencehIL-1lp, e.g. native sequence hIL-1Ra1 or hIL-1Ra3.

In another embodiment, the invention provides a method for treating anischemic reperfusion disease, such as surgical tissue reperfusioninjury, stroke, myocardial ischemia, or acute myocardial infarction,comprising administering to a mammal, such as human, in need of suchtreatment an effective amount of an hIL-1Ra1L, such as a native sequencehIL-1Ra1L, or an effective amount of an hIL-1Ra1V, such as a nativesequence hIL-1Ra1V, or an effective amount of an hIL-1Ra1S, such as anative sequence hIL-1Ra1S.

In another embodiment, the invention provides a method for treatingpsoriasis comprising administering to a mammal, such as human, in needof such treatment an effective amount of an IL-1lp, such as a nativesequence IL-1lp.

In another embodiment, the invention provides a method for treatingpsoriasis comprising administering to a mammal, such as human, in needof such treatment an effective amount of an IL-1lp selected from thegroup consisting of hIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2,hIL-1Ra3, and mIL-1Ra3.

In another embodiment, the invention provides a method for treatingpsoriasis comprising administering to a mammal, such as human, in needof such treatment an effective amount of an hIL-1lp, such as a nativesequence hIL-1lp, e.g. native sequence hIL-1Ra1 or hIL-1Ra3.

In another embodiment, the invention provides a method for treatingpsoriasis comprising administering to a mammal, such as human, in needof such treatment an effective amount of an hIL-1Ra1L, such as a nativesequence hIL-1Ra1L, or an effective amount of an hIL-1Ra1V, such as anative sequence hIL-1Ra1V, or an effective amount of an hIL-1Ra1S, suchas a native sequence hIL-1Ra1S.

In another embodiment, the invention provides a method for treatinggraft-versus-host disease (GVHD) comprising administering to a mammal,such as human, in need of such treatment an effective amount of anIL-1lp, such as a native sequence IL-1lp.

In another embodiment, the invention provides a method for treatinggraft-versus-host disease (GVHD) comprising administering to a mammal,such as human, in need of such treatment an effective amount of anIL-1lp selected from the group consisting of hIL-1Ra1, hIL-1Ra1L,hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, and mIL-1Ra3.

In another embodiment, the invention provides a method for treatinggraft-versus-host disease (GVHD) comprising administering to a mammal,such as human, in need of such treatment an effective amount of anhIL-1lp, such as a native sequence hIL-1lp, e.g. native sequencehIL-1Ra1 or hIL-1Ra3.

In another embodiment, the invention provides a method for treatinggraft-versus-host disease (GVHD) comprising administering to a mammal,such as human, in need of such treatment an effective amount of anhIL-1Ra1L, such as a native sequence hIL-1Ra1L, or an effective amountof an hIL-1Ra1V, such as a native sequence hIL-1Ra1V, or an effectiveamount of an hIL-1Ra1S, such as a native sequence hIL-1Ra1S.

In another embodiment, the invention provides a method for treating aninflammatory bowel disease such as ulcerative colitis, comprisingadministering to a mammal, such as human, in need of such treatment aneffective amount of an IL-1lp, such as a native sequence IL-1lp.

In another embodiment, the invention provides a method for treating aninflammatory bowel disease such as ulcerative colitis, comprisingadministering to a mammal, such as human, in need of such treatment aneffective amount of an IL-1lp selected from the group consisting ofhIL-1Ra1, hIL-1Ra1L, hIL-1Ra1V, hIL-1Ra1S, hIL-1Ra2, hIL-1Ra3, andmIL-1Ra3.

In another embodiment, the invention provides a method for treating aninflammatory bowel disease such as ulcerative colitis, comprisingadministering to a mammal, such as human, in need of such treatment aneffective amount of an hIL-1lp, such as a native sequence hIL-1lp, e.g.native sequence hIL-1Ra1 or hIL-1Ra3.

In another embodiment, the invention provides a method for treating aninflammatory bowel disease such as ulcerative colitis, comprisingadministering to a mammal, such as human, in need of such treatment aneffective amount of an hIL-1Ra1L, such as a native sequence hIL-1Ra1L,or an effective amount of an hIL-1Ra1V, such as a native sequencehIL-1Ra1V, or an effective amount of an hIL-1Ra1S, such as a nativesequence hIL-1Ra1S.

D. Anti-IL-1lp Antibodies

The present invention further provides anti-IL-1lp antibodies. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-IL-1lp antibodies may comprise polyclonal antibodies. Methodsof preparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the IL-1lp polypeptide or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-IL-1lp antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the IL-1lp polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed againstIL-1lp. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunosorbant assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Human and Humanized Antibodies

The anti-IL-1lp antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321: 522-525 (1986); Riechmann etal., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2: 593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature,332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)],by substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1): 86-95 (1991)].Similarly, human antibodies can be made by introducing of humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51(1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg andHuszar, Intern. Rev. Immunol. 13 65-93 (1995).

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe IL-1lp, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10: 3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

E. Uses for anti-IL-1lp Antibodies

The anti-IL-1lp antibodies of the invention have various utilities. Forexample, anti-IL-1lp antibodies may be used in diagnostic assays forIL-1lp, e.g., detecting its expression in specific cells, tissues, orserum. Various diagnostic assay techniques known in the art may be used,such as competitive binding assays, direct or indirect sandwich assaysand immunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144: 945 (1962); David et al., Biochemistry, 13: 1014 (1974); Pain etal., J. Immunol. Meth., 40: 219 (1981); and Nygren, J. Histochem. andCytochem., 30: 407 (1982).

Anti-IL-1lp antibodies also are useful for the affinity purification ofIL-1lp from recombinant cell culture or natural sources. In thisprocess, the antibodies against IL-1lp are immobilized on a suitablesupport, such a Sephadex resin or filter paper, using methods well knownin the art. The immobilized antibody then is contacted with a samplecontaining the IL-1lp to be purified, and thereafter the support iswashed with a suitable solvent that will remove substantially all thematerial in the sample except the IL-1lp, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent that will release the IL-1lp from the antibody.

In addition, anti-IL-1lp antibodies are useful as therapeutic agents fortargeting of native IL-1lp in IL-1lp-mediated disease conditions, e.g.disease states characterized by pathologic IL-1 or IL-18 agonist oragonist-like activity of the native IL-1lp. In the treatment andprevention of a native IL-1lp-mediated disorder with the anti-IL-1lpantibody of the invention, the antibody composition will be formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the antibody, the particular type ofantibody, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“effective amount” or “therapeutically effective amount” of antibody tobe administered will be governed by such considerations, and is theminimum amount necessary to prevent, ameliorate, or treat the nativeIL-1lp-mediated disorder, including treating inflammatory diseases andreducing inflammatory responses. Such amount is preferably below theamount that is toxic to the host or renders the host significantly moresusceptible to infections.

As a general proposition, the initial pharmaceutically effective amountof the antibody or antibody fragment administered parenterally per dosewill be in the range of about 0.1 to 50 mg/kg of patient body weight perday, with the typical initial range of antibody used being 0.3 to 20mg/kg/day, more preferably 0.3 to 15 mg/kg/day.

In one embodiment, using systemic administration, the initialpharmaceutically effective amount will be in the range of about 2 to 5mg/kg/day.

For methods of the invention using administration by inhalation, theinitial pharmaceutically effective amount will be in the range of about1 microgram (μg)/kg/day to 100 mg/kg/day.

In one embodiment, the invention provides a method for treating anIL-1lp-mediated inflammatory disorder comprising administering to amammal, such as human, in need of such treatment an effective amount ofan anti-IL-1lp antibody.

In another embodiment, the invention provides a method for treating anhIL-1lp-mediated asthmatic disorder comprising administering to a humanin need of such treatment an effective amount of an anti-hIL-1lpantibody.

In another embodiment, the invention provides a method for treating anhIL-1Ra1-mediated asthmatic disorder comprising administering to a humanin need of such treatment an effective amount of an anti-hIL-1Ra1antibody.

In another embodiment, the invention provides a method for treating anIL-1lp-mediated rheumatoid arthritic disorder comprising administeringto a mammal, such as human, in need of such treatment an effectiveamount of an anti-IL-1lp antibody.

In another embodiment, the invention provides a method for treating anhIL-1lp-mediated rheumatoid arthritic disorder comprising administeringto a human in need of such treatment an effective amount of ananti-hIL-1lp antibody.

In another embodiment, the invention provides a method for treating anhIL-1Ra1-mediated rheumatoid arthritic disorder comprising administeringto a human in need of such treatment an effective amount of ananti-hIL-1Ra1 antibody.

In another embodiment, the invention provides a method for treating anIL-1lp-mediated osteoarthritic disorder comprising administering to amammal, such as human, in need of such treatment an effective amount ofan anti-IL-1lp antibody.

In another embodiment, the invention provides a method for treating anhIL-1lp-mediated osteoarthritic disorder comprising administering to ahuman in need of such treatment an effective amount of an anti-hIL-1lpantibody.

In another embodiment, the invention provides a method for treating anhIL-1Ra1-mediated osteoarthritic disorder comprising administering to ahuman in need of such treatment an effective amount of an anti-hIL-1Ra1antibody.

In another embodiment, the invention provides a method for treating anIL-1lp-mediated septic disorder comprising administering to a mammal,such as human, in need of such treatment an effective amount of ananti-IL-1lp antibody.

In another embodiment, the invention provides a method for treating anhIL-1lp-mediated septic disorder comprising administering to a human inneed of such treatment an effective amount of an anti-hIL-1lp antibody.

In another embodiment, the invention provides a method for treating anhIL-1Ra1-mediated septic disorder comprising administering to a human inneed of such treatment an effective amount of an anti-hIL-1Ra1 antibody.

In another embodiment, the invention provides a method for treatingIL-1lp-mediated acute lung injury comprising administering to a mammal,such as human, in need of such treatment an effective amount of ananti-IL-1lp antibody.

In another embodiment, the invention provides a method for treatinghIL-1lp-mediated acute lung injury comprising administering to a humanin need of such treatment an effective amount of an anti-hIL-1lpantibody.

In another embodiment, the invention provides a method for treatinghIL-1Ra1-mediated acute lung injury comprising administering to a humanin need of such treatment an effective amount of an anti-hIL-1Ra1antibody.

In another embodiment, the invention provides a method for treatingIL-1lp-mediated adult respiratory distress syndrome comprisingadministering to a mammal, such as human, in need of such treatment aneffective amount of an anti-IL-1lp antibody.

In another embodiment, the invention provides a method for treatinghIL-1lp-mediated adult respiratory distress syndrome comprisingadministering to a human in need of such treatment an effective amountof an anti-hIL-1lp antibody.

In another embodiment, the invention provides a method for treatinghIL-1Ra1-mediated adult respiratory distress syndrome comprisingadministering to a human in need of such treatment an effective amountof an anti-hIL-1Ra1 antibody.

In another embodiment, the invention provides a method for treatingIL-1lp-mediated idiopathic pulmonary fibrosis comprising administeringto a mammal, such as human, in need of such treatment an effectiveamount of an anti-IL-1lp antibody.

In another embodiment, the invention provides a method for treatinghIL-1lp-mediated idiopathic pulmonary fibrosis comprising administeringto a human in need of such treatment an effective amount of ananti-hIL-1lp antibody.

In another embodiment, the invention provides a method for treatinghIL-1Ra1-mediated idiopathic pulmonary fibrosis comprising administeringto a human in need of such treatment an effective amount of ananti-hIL-1Ra1 antibody.

In another embodiment, the invention provides a method for treating anIL-1lp-mediated ischemic reperfusion disease, such as surgical tissuereperfusion injury, stroke, myocardial ischemia, or acute myocardialinfarction, comprising administering to a mammal, such as human, in needof such treatment an effective amount of an anti-IL-1lp antibody.

In another embodiment, the invention provides a method for treating anhIL-1lp-mediated ischemic reperfusion disease, such as surgical tissuereperfusion injury, stroke, myocardial ischemia, or acute myocardialinfarction, comprising administering to a human in need of suchtreatment an effective amount of an anti-hIL-1lp antibody.

In another embodiment, the invention provides a method for treating anhIL-1Ra1-mediated ischemic reperfusion disease, such as surgical tissuereperfusion injury, stroke, myocardial ischemia, or acute myocardialinfarction, comprising administering to a human in need of suchtreatment an effective amount of an anti-hIL-1Ra1 antibody.

In another embodiment, the invention provides a method for treating anIL-1lp-mediated psoriatic disorder comprising administering to a mammal,such as human, in need of such treatment an effective amount of ananti-IL-1lp antibody.

In another embodiment, the invention provides a method for treating anhIL-1lp-mediated psoriatic disorder comprising administering to a humanin need of such treatment an effective amount of an anti-hIL-1lpantibody.

In another embodiment, the invention provides a method for treating anhIL-1Ra1-mediated psoriatic disorder comprising administering to a humanin need of such treatment an effective amount of an anti-hIL-1Ra1antibody.

In another embodiment, the invention provides a method for treating anIL-1lp-mediated graft-versus-host disease (GVHD) comprisingadministering to a mammal, such as human, in need of such treatment aneffective amount of an anti-IL-1lp antibody.

In another embodiment, the invention provides a method for treating anhIL-1lp-mediated graft-versus-host disease (GVHD) comprisingadministering to a human in need of such treatment an effective amountof an anti-hIL-1lp antibody.

In another embodiment, the invention provides a method for treating anhIL-1Ra1-mediated graft-versus-host disease (GVHD) comprisingadministering to a human in need of such treatment an effective amountof an anti-hIL-1Ra1 antibody.

In another embodiment, the invention provides a method for treating anIL-1lp-mediated inflammatory bowel disease such as ulcerative colitis,comprising administering to a mammal, such as human, in need of suchtreatment an effective amount of an anti-IL-1lp antibody.

In another embodiment, the invention provides a method for treating anhIL-1lp-mediated inflammatory bowel disease such as ulcerative colitis,comprising administering to a human in need of such treatment aneffective amount of an anti-hIL-1lp antibody.

In another embodiment, the invention provides a method for treating anhIL-1Ra1-mediated inflammatory bowel disease such as ulcerative colitis,comprising administering to a human in need of such treatment aneffective amount of an anti-hIL-1Ra1 antibody.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Isolation of DNA encoding hIL-1Ra1 and mIL-1Ra3

A public expressed sequence tag (EST) DNA database (Genbank) wassearched with human interleukin-1 receptor antagonist (hIL-1Ra)sequence, also known as secretory human interleukin-1 receptorantagonist (“sIL-1Ra”) sequence, and a human EST designated AI014548(FIG. 4, SEQ ID NO:8), and a murine EST designated W08205 (FIG. 10, SEQID NO:17), were identified, which showed homology with the known proteinhIL-1Ra (sIL-1Ra).

EST clones AI014548 and W08205 were purchased from Research Genetics(Huntsville, Ala.) and the cDNA inserts were obtained and sequenced intheir entireties.

The entire nucleotide sequence of the clone AI014548, designatedDNA85066, is shown in FIG. 1 (SEQ ID NO:1). Clone DNA85066 contains asingle open reading frame that is interrupted by an apparent intronicsequence. The intron is bounded by splice junctions at nucleotidepositions 181 to 186 (splice donor site) and nucleotide positions 430 to432 (splice acceptor site) (FIG. 1; SEQ ID NO:1).

A virtual processed nucleotide sequence (FIG. 3; SEQ ID NO:6),designated DNA94618, was derived by removing the apparent intronicsequence from clone DNA85066. Clone DNA94618 contains a single openreading frame with an apparent translational initiation site atnucleotide positions 103-105, and a stop codon at nucleotide positions682-684 (FIG. 3; SEQ ID NO:6). The predicted polypeptide precursor(hIL-1Ra1) (FIG. 3; SEQ ID NO:7) is 193 amino acids long. The putativesignal sequence extends from amino acid positions 1 to 14. A putativecAMP- and cGMP-dependent protein kinase phosphorylation site is locatedat amino acid positions 33-36. Putative N-myristoylation sites arelocated at amino acid positions 50-55 and 87-92.

Clone DNA85066 (designated as DNA85066-2534) has been deposited withATCC and was assigned ATCC deposit no. 203588. The full-length hIL-1Ra1protein shown in FIG. 3 (SEQ ID NO:7) has an estimated molecular weightof about 21,822 daltons and a pI of about 8.9.

Based on a sequence alignment analysis of the full-length sequence (SEQID NO:7), hIL-1Ra1 shows significant amino acid sequence identity tohIL-1Ra (sIL-1Ra) and hIL-1Raβ proteins.

The entire nucleotide sequence of the clone W08205, designated DNA92505,is shown in FIG. 9 (SEQ ID NO:15). Clone DNA92505 contains a single openreading frame with an apparent translational initiation site atnucleotide positions 145-147, and a stop codon at nucleotide positions610-612 (FIG. 9; SEQ ID NO:15). The predicted polypeptide precursor(mIL-1Ra3) (FIG. 9; SEQ ID NO:16) is 155 amino acids long. The putativesignal sequence extends from amino acid positions 1-33. PutativeN-myristoylation sites are located at amino acid positions 29-34, 60-65,63-68, 91-96 and 106-111. An interleukin-1-like sequence is located atamino acid positions 111-131.

Clone DNA92505 (designated as DNA92505-2534) was deposited with ATCC andwas assigned ATCC deposit no. 203590. The full length mIL-1Ra3 proteinshown in FIG. 9 (SEQ ID NO:16) has an estimated molecular weight ofabout 17,134 daltons and a pI of about 4.8.

Based on a sequence alignment analysis of the full-length sequence (SEQID NO:16), mIL-1Ra3 shows significant amino acid sequence identity tomIL-1Ra, hicIL-1Ra, hIL-1Ra (sIL-1Ra) and hIL-1Raβ proteins.

Example 2 Isolation of DNA encoding hIL-1ra2 and hIL-1Ra3

A expressed sequence tag (EST) DNA database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.) was searched with humaninterleukin-1 receptor antagonist (hIL-1Ra) sequence, also known assecretory human interleukin-1 receptor antagonist (“sIL-1Ra”) sequence,and the ESTs, designated 1433156 (FIG. 5, SEQ ID NO:9) and 5120028 (FIG.7, SEQ ID NO:12), were identified, which showed homology with thehIL-1Ra known protein.

EST clones 1433156 and 5120028 were purchased from IncytePharmaceuticals (Palo Alto, Calif.) and the cDNA inserts were obtainedand sequenced in their entireties.

The entire nucleotide sequence of the clone 1433156, designatedDNA92929, is shown in FIG. 5 (SEQ ID NO:9). Clone DNA92929 contains asingle open reading frame with an apparent translational initiation siteat nucleotide positions 96-98, and a stop codon at nucleotide positions498-500 (FIG. 5; SEQ ID NO:9). The predicted polypeptide precursor(hIL-1Ra2) (FIG. 5; SEQ ID NO:10) is 134 amino acids long. A putativesignal sequence extends from amino acid positions 1-26.

Clone DNA92929 (designated as DNA92929-2534) was deposited with ATCC andwas assigned ATCC deposit no. 203586. The full-length hIL-1Ra2 proteinshown in FIG. 5 (SEQ ID NO:10) has an estimated molecular weight ofabout 14,927 daltons and a pI of about 4.8.

Based on a sequence alignment analysis of the full-length sequence (SEQID NO:10), hIL-1Ra2 shows significant amino acid sequence identity tohIL-1Raβ protein. hIL-1Ra2 is believed to be a splice variant ofhIL-1Raβ.

The entire nucleotide sequence of the clone 5120028, designatedDNA96787, is shown in FIG. 7 (SEQ ID NO:12). Clone DNA96787 contains asingle open reading frame with an apparent translational initiation siteat nucleotide positions 1-3, and a stop codon at nucleotide positions466-468 (FIG. 7; SEQ ID NO:12). The predicted polypeptide precursor(hIL-1Ra3) (FIG. 7; SEQ ID NO:13) is 155 amino acids long. A putativesignal sequence extends from amino acid positions 1-33. PutativeN-myristoylation sites are located at amino acid positions 29-34, 60-65,63-68, 73-78, 91-96 and 106-111. An interleukin-1-like sequence islocated at amino acid positions 111-131.

It is believed that the predicted 155 amino acid polypeptide of hIL-1Ra3behaves as a mature sequence (without a presequence that is removed inpost-translational processing) in certain animal cells. It is alsobelieved that other animal cells recognize and remove one or more signalpeptide(s) extending from amino acid positions 1 to about 33. As shownin Example 14 below, transiently transfected CHO host cells secrete aform of hIL-1Ra3 that only lacks the N-terminal methionine in thesequence of FIG. 7 (SEQ ID NO:13).

Clone DNA96787 (designated as DNA96787-2534) was deposited with ATCC andwas assigned ATCC deposit no. 203589. The full length hIL-1Ra3 proteinshown in FIG. 7 (SEQ ID NO:13) has an estimated molecular weight ofabout 16,961 daltons and a pI of about 4.9.

Based on a sequence alignment analysis of the full-length sequence (SEQID NO:13), hIL-1Ra3 shows significant amino acid sequence identity tohicIL-1Ra and hIL-1Ra (sIL-1Ra) proteins.

Example 3 Northern Blot Analysis

Expression of hIL-1Ra3 mRNA in human tissues and mIL-1Ra3 mRNA in mousetissues was examined by Northern blot analysis. Human and mouse multipletissue northern (RNA) blots and mouse embryo blots were purchased fromClontech and probed with corresponding cDNA according to themanufacturer's instructions.

As shown in FIG. 11, hIL-1Ra3 mRNA (2.7 kb) were detected only in humanplacenta and mIL-1Ra3 mRNA transcripts (1.4 kb and 2.5 kb) were detectedonly in the day-17 mouse embryo.

Example 4 Use of IL-1lp as a Hybridization Probe

The following method describes use of a nucleotide sequence encodingIL-1lp as a hybridization probe.

DNA comprising the coding sequence of full-length IL-1lp (as shown inFIGS. 3, 5, 7, 9, 15, 16 and 19; SEQ ID NOS:6, 9, 12, 15, 18, 20 and 24)is employed as a probe to screen for homologous DNAs (such as thoseencoding naturally-occurring variants of IL-1lp) in human tissue cDNAlibraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled IL-1lp-derived probe to the filters is performed in asolution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate,50 mM sodium phosphate, pH 6.8, 2× Denhardt's solution, and 10% dextransulfate at 42° C. for 20 hours. Washing of the filters is performed inan aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence IL-1lp can then be identified using standardtechniques known in the art.

Example 5 Expression of IL-1lp in E. coli

This example illustrates preparation of an unglycosylated form of IL-1lpby recombinant expression in E. coli.

The DNA sequence encoding an IL-1lp is initially amplified usingselected PCR primers. The primers should contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector. A variety of expression vectors may be employed. Anexample of a suitable vector is pBR322 (derived from E. coli; seeBolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillinand tetracycline resistance. The vector is digested with restrictionenzyme and dephosphorylated. The PCR amplified sequences are thenligated into the vector. The vector will preferably include sequenceswhich encode for an antibiotic resistance gene, a trp promoter, apolyhis leader (including the first six STII codons, polyhis sequence,and enterokinase cleavage site), the IL-1lp coding region, lambdatranscriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized IL-1lp protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

Example 6 Expression of IL-1lp in Mammalian Cells

This example illustrates preparation of a potentially glycosylated formof IL-1lp by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the IL-1lp DNA is ligated intopRK5 with selected restriction enzymes to allow insertion of the IL-1lpDNA using ligation methods such as described in Sambrook et al., supra.The resulting vector is called pRK5-IL-1lp.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μgpRK5-IL-1lp DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of IL-1lp polypeptide. The cultures containing transfectedcells may undergo further incubation (in serum free medium) and themedium is tested in selected bioassays.

In an alternative technique, IL-1lp may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci. USA, 12: 7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-IL-1lp DNA is added.The cells are first concentrated from the spinner flask bycentrifugation and washed with PBS. The DNA-dextran precipitate isincubated on the cell pellet for four hours. The cells are treated with20% glycerol for 90 seconds, washed with tissue culture medium, andre-introduced into the spinner flask containing tissue culture medium, 5μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about fourdays, the conditioned media is centrifuged and filtered to remove cellsand debris. The sample containing expressed IL-1lp can then beconcentrated and purified by any selected method, such as dialysisand/or column chromatography.

In another embodiment, IL-1lp can be expressed in CHO cells. ThepRK5-IL-1lp can be transfected into CHO cells using known reagents suchas CaPO₄ or DEAE-dextran. As described above, the cell cultures can beincubated, and the medium replaced with culture medium (alone) or mediumcontaining a radiolabel such as ³⁵S-methionine. After determining thepresence of IL-1lp polypeptide, the culture medium may be replaced withserum free medium. Preferably, the cultures are incubated for about 6days, and then the conditioned medium is harvested. The mediumcontaining the expressed IL-1lp can then be concentrated and purified byany selected method.

Epitope-tagged IL-1lp may also be expressed in host CHO cells. TheIL-1lp may be subcloned out of the pRK5 vector. The subclone insert canundergo PCR to fuse in frame with a selected epitope tag such as apoly-his tag into a Baculovirus expression vector. The poly-his taggedIL-1lp insert can then be subcloned into a SV40 driven vector containinga selection marker such as DHFR for selection of stable clones. Finally,the CHO cells can be transfected (as described above) with the SV40driven vector. Labeling may be performed, as described above, to verifyexpression. The culture medium containing the expressed poly-His taggedIL-1lp can then be concentrated and purified by any selected method,such as by Ni²⁺-chelate affinity chromatography.

Example 7 Expression of IL-1lp in Yeast

The following method describes recombinant expression of IL-1lp inyeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of IL-1lp from the ADH2/GAPDH promoter. DNAencoding IL-1lp and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof IL-1lp. For secretion, DNA encoding IL-1lp can be cloned into theselected plasmid, together with DNA encoding the ADH2/GAPDH promoter, anative IL-1lp signal peptide or other mammalian signal peptide, or, forexample, a yeast alpha-factor or invertase secretory signal/leadersequence, and linker sequences (if needed) for expression of IL-1lp.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant IL-1lp can subsequently be isolated and purified by removingthe yeast cells from the fermentation medium by centrifugation and thenconcentrating the medium using selected cartridge filters. Theconcentrate containing IL-1lp may further be purified using selectedcolumn chromatography resins.

Example 8 Expression of IL-1lp in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of IL-1lp inBaculovirus-infected insect cells.

The sequence coding for IL-1lp is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding IL-1lp or the desired portion of the coding sequenceof IL-1lp (such as the sequence encoding the mature protein) isamplified by PCR with primers complementary to the 5′ and 3′ regions.The 5′ primer may incorporate flanking (selected) restriction enzymesites. The product is then digested with those selected restrictionenzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged IL-1lp can then be purified, for example, byNi²⁺-chelate affinity chromatography as follows. Extracts are preparedfrom recombinant virus-infected Sf9 cells as described by Rupert et al.,Nature, 362: 175-179 (1993). Briefly, Sf9 cells are washed, resuspendedin sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA;10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 secondson ice. The sonicates are cleared by centrifugation, and the supernatantis diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM Imidazole gradient in the secondary wash buffer. One mL fractionsare collected and analyzed by SDS-PAGE and silver staining or Westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted His₁₀-tagged IL-1lp are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) IL-1lp canbe performed using known chromatography techniques, including forinstance, Protein A or protein G column chromatography.

Example 9 IL-18 Receptor and IL-1 Receptor Binding of hIL-1Ra1

To facilitate the characterization of hIL-1Ra1, a PCR fragmentcontaining the partial ORF of clone DNA85066 (FIG. 1; SEQ ID NO:3) wascloned into pCMV1FLAG (IBI Kodak, described in Pan et al., Science, 276:111-113) as an in-frame fusion to a NH₂-terminal preprotrypsin leadersequence and FLAG tag encoded by the vector. The entire cDNA insert ofthe recombinant pCMV1FLAG vector clone (designated clone DNA96786) wassequenced (FIG. 2; SEQ ID NO:4). The cDNAs encoding the extracellulardomain of hIL1R and hIL18R (formerly known as hIL1Rrp) were obtained bypolymerase chain reaction (PCR) and cloned into a modified pCMV1FLAGvector that allowed for in-frame fusion with the Fc portion of humanimmunoglobulin G.

Human embryonic kidney 293 cells were grown in high glucose DMEM(Genentech, Inc). The cells were seeded at 3-4×10⁶ per plate (100 mm)and co-transfected with pCMV 1FLAG-hIL-1Ra1 and pCMV1FLAG-IL1R-ECD-Fc orpCMV1FLAG-IL18R-ECD-Fc by means of calcium phosphate precipitation. Themedia were changed 12 hours post transfection. The resultant conditionedmedia (10 ml each) were harvested after a further 70-74 hour incubation,clarified by centrifugation, aliquoted and stored at −70° C. Thereceptor-Fc and ligand complex from 1.5 ml conditioned medium wasimmunoprecipitated with protein G-Sepharose, washed three times withbuffer containing 50 mM Hepes, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40,and a protease inhibitor cocktail (BMB) and resolved on a 10-20%SDS-PAGE gel. The bound ligand was identified by immunoblotting usinganti-FLAG monoclonal antibody (BMB).

As shown in FIG. 13A, the secreted FLAGhIL-1Ra1 fusion protein bound toIL-18R ECD and did not bind to IL-1R ECD, which indicates that hIL-1Ra1could be an agonist or antagonist of IL-18R.

Example 10 IL-1 Receptor and IL-18 Receptor Binding of mIL-1Ra3 andhIL-1Ra3

cDNA encoding mIL-1Ra3 (DNA92505 shown in FIG. 9; SEQ ID NO:15) wascloned into pRK7 with a carboxy-terminal FLAG-tag. The resultingexpression construct was transfected into human embryonic kidney 293cells by means of calcium phosphate precipitation. 84-90 hours posttransfection, the conditioned media containing secreted FLAGmIL-1Ra3fusion protein was harvested. Conditioned media containing secretedIL-18R-Fc and IL-1R-Fc proteins were prepared as described in Example 9above, with the exception that the 293 cells were transfected witheither pCMV1FLAG-IL1R-ECD-Fc or pCMV1FLAG-IL18R-ECD-Fc alone (withoutpCMV1FLAG-IL-1Ra1 cotransfection).

For in vitro binding assays, IL-1R-Fc or IL-18R-Fc from 0.5 ml of theconditioned medium was immobilized to protein G-agarose and then mixedwith 1.2 ml conditioned medium containing FLAGmIL-1Ra3. Thereceptor-ligand complexes were washed and resolved on an 10-20% SDS-PAGEgel and the bound ligand was detected by immunoblotting using anti-FLAGmonoclonal antibody (Boehringer Mannheim).

As shown in FIG. 14, FLAGmIL-1Ra3 fusion protein bound to IL-1R ECD anddid not bind to IL-18R ECD. Since the amino acid sequence of mIL-1Ra3 isrelated to that of the known interleukin-1 receptor antagonist protein(IL-1Ra), mIL-3Ra3 is believed to be a novel IL-1 receptor antagonist.

cDNA encoding hIL-1Ra3 (DNA96787 shown in FIG. 7; SEQ ID NO:12) wascloned into pRK7 with a carboxy-terminal FLAG tag to formpRK7hIL-1Ra3-FLAG. pCMV1FLAG-IL1R-ECD-Fc and pCMV1FLAG-IL18R-ECD-Fc wereobtained as described in Example 9 above. Similarly, cDNA encoding DR6was cloned into the modified pCMV1FLAG vector of Example 9 to formpCMV1FLAG-DR6-Fc, encoding DR6 fused to the Fc portion of humanimmunoglobulin G. Conditioned media containing (1) a combination ofsecreted FLAGhIL-1Ra3 and FLAG-DR6-Fc (2) a combination of secretedFLAGhIL-1Ra3 and FLAG-IL1R-ECD-Fc or (3) a combination of secretedFLAGhIL-1Ra3 and FLAG-IL18R-ECD-Fc were prepared by cotransfecting Human293 cells with (1) pRK7hIL-1Ra3-FLAG and pCMV1FLAG-DR6-Fc (2)pRK7hIL-1Ra3-FLAG and pCMV1FLAG-IL1R-ECD-Fc or (3) pRK7hIL-1Ra3-FLAG andpCMV1FLAG-IL18R-ECD-Fc, culturing the transfectant cells and harvestingculture media essentially as described in Example 9 above. Thereceptor-Fc and ligand complex from each conditioned medium wasimmunoprecipitated with protein G-Sepharose or anti-FLAG monoclonalantibody, and immunoprecipitates were resolved by gel electrophoresisand immunoblotting with anti-FLAG monoclonal antibody essentially asdescribed in Example 9 above.

As shown in FIG. 13B, FLAGhIL-1Ra3 fusion protein bound to IL-1R-ECD-Fcand did not bind to IL-18R-ECD-Fc or DR6-Fc. Since the amino acidsequence of hIL-1Ra3 is related to that of the known interleukin-1receptor antagonist protein (IL-1Ra), hIL-3Ra3 is believed to be a novelIL-1 receptor antagonist.

Example 11 Preparation of Antibodies that Bind IL-1lp

This example illustrates preparation of monoclonal antibodies which canspecifically bind IL-1lp.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified IL-1lp, fusion proteins containing IL-1lp,and cells expressing recombinant IL-1lp on the cell surface. Selectionof the immunogen can be made by the skilled artisan without undueexperimentation.

Mice, such as Balb/c, are immunized with the IL-1lp immunogen emulsifiedin complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-IL-1lp antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of IL-1lp. Three to four days later, the mice are sacrificedand the spleen cells are harvested. The spleen cells are then fused(using 35% polyethylene glycol) to a selected murine myeloma cell linesuch as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusionsgenerate hybridoma cells which can then be plated in 96 well tissueculture plates containing HAT (hypoxanthine, aminopterin, and thymidine)medium to inhibit proliferation of non-fused cells, myeloma hybrids, andspleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstIL-1lp. Determination of “positive” hybridoma cells secreting thedesired monoclonal antibodies against IL-1lp is within the skill in theart.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-IL-1lpmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 12 Isolation of DNA encoding hIL-1Ra1L, hIL-1Ra1V and hIL-1Ra1S

Several intron-containing cDNA clones related to the hIL-1Ra1intron-containing clone DNA85066 (FIG. 2) (SEQ ID NO:4) were isolatedfrom a human testis cDNA library and fully sequenced. Theintron-containing cDNA sequences were used to determine a full-lengthopen reading frame (ORF) with the GENESCAN program(http://CCR-081.mit.edu/GENESCAN.html). The ORF-encoding sequence wasused to design two DNA primers, ggc gga tcc aaa atg ggc tct gag gac tggg (SEQ ID NO:29) (1Ra1016) and gcg gaa ttc taa tcg ctg acc tca ctg ggg(SEQ ID NO:30) (1Ra1017). The 1Ra1016 and 1Ra1017 primers weresynthesized and used to clone cDNA from human fetal skin and SK-lu-1cell cDNA libraries using polymerase chain reaction (PCR). Several PCRproducts were isolated and sequenced. Two full length cDNA clones(designated DNA102043 and DNA 102044) from PCR products were found toencode hIL-1Ra1 isoforms.

The entire nucleotide sequence of clone DNA102043 is shown in FIG. 15(SEQ ID NO:18). Clone DNA102043 contains a single open reading framewith an apparent translational initiation site at nucleotide positions4-6, and a stop codon at nucleotide positions 625-627 (FIG. 15; SEQ IDNO:18). The predicted polypeptide precursor (designated hIL-1Ra1L) (FIG.15; SEQ ID NO:19) is 207 amino acids long. The putative signal sequenceextends from amino acid positions 1-34.

Clone DNA102043 (designated DNA102043-2534) was deposited with ATCC andwas assigned ATCC deposit no. 203846. The full-length hIL-1Ra1L proteinshown in FIG. 15 (SEQ ID NO:19) has an estimated molecular weight ofabout 23,000 daltons and a pI of about 6.08.

Based on a sequence alignment analysis of the full length sequence (SEQID NO:19), hIL-1Ra1L shows significant amino acid sequence identity tohIL-1Raβ and TANGO-77 protein. In addition, a portion of the DNAsequence of clone DNA102043 (FIG. 15) (SEQ ID NO:18) was found tocoincide with the DNA sequence of EST AI014548 (FIG. 4) (SEQ ID NO:8)and with the complement of the DNA sequence of EST AI323258 (FIG. 17)(SEQ ID NO:23).

The entire nucleotide sequence of clone DNA102044 is shown in FIG. 16(SEQ ID NO:20). Clone DNA102044 contains a single open reading framewith an apparent translational initiation site at nucleotide positions4-6, and a stop codon at nucleotide positions 505-507 (FIG. 16; SEQ IDNO:20). The predicted polypeptide (designated hIL-1Ra1S) (FIG. 16; SEQID NO:21) is 167 amino acids long, and it is believed to behave as amature sequence (without a presequence that is removed inpost-translational processing) in certain animal cells. In addition, itis believed that other animal cells recognize and remove inpost-translational processing one or more signal peptide(s) contained inthe sequence extending from amino acid positions 1 to about 46.

Clone DNA102044 (designated DNA102044-2534) was deposited with ATCC andwas assigned ATCC deposit no. 203855. The full-length hIL-1Ra1S proteinshown in FIG. 16 (SEQ ID NO:21) has an estimated molecular weight ofabout 18,478 daltons and a pI of about 5.5.

Based on a sequence alignment analysis of the full length sequence (SEQID NO:21), hIL-1Ra1S appears to be an allelic variant of TANGO-77protein and also shows significant amino acid sequence identity tohIL-1Raβ. In addition, a portion of the DNA sequence of clone DNA102044(FIG. 16) (SEQ ID NO:20) was found to coincide with the DNA sequence ofEST AI014548 (FIG. 4) (SEQ ID NO:8) and with the complement of the DNAsequence of EST AI323258 (FIG. 17) (SEQ ID NO:23).

EST clone AI323258 was purchased from Research Genetics (Huntsville,Ala.) and the cDNA insert was obtained and sequenced in its entirety.The entire sequence of the clone AI323258, designated DNA114876, isshown in FIG. 19 (SEQ ID NO:24). Clone DNA114876 contains a single openreading frame (ORF) with an apparent translation initiation site atnucleotide positions 73-75 and a stop codon at nucleotide positions726-728 (FIG. 19; SEQ ID NO:24), encoding a predicted polypeptideprecursor (hIL-1Ra1V) (FIG. 19; SEQ ID NO:25) that is 218 amino acidslong. In addition, the ORF contains an alternate translation initiationsite at nucleotide positions 106-108. The predicted polypeptide (alsodesignated hIL-1Ra1V) for translation initiated at the alternate startcodon is 207 amino acids in length (lacking the first eleven residues atthe N-terminus of the 218 amino acid polypeptide). It is believed thatthe predicted 218 amino acid and 207 amino acid polypeptides behave asmature sequences (without a presequence that is removed inpost-translational processing) in certain animal cells. It is alsobelieved that other animal cells recognize and remove one or more signalpeptide(s) extending from amino acid positions 1 to about 48 (a putativeleader sequence in the 218 amino acid polypeptide) or from amino acidpositions 12 to 36 (a putative leader sequence in the 207 amino acidpolypeptide) in the amino acid sequence of FIG. 19 (SEQ ID NO:25). Asshown in Example 14 below, transiently transfected CHO host cellssecrete unprocessed forms of hIL-1Ra1V and hIL-1Ra1L and a singleprocessed form that results from the removal of a signal peptideextending from amino acid positions 1 to 45 in FIG. 19 (SEQ ID NO:25) orthe removal of a signal peptide extending from amino acid positions 1 to34 of FIG. 15 (SEQ ID NO:19). The processed form of hIL-1Ra1V andhIL-1Ra1L secreted by transiently transfected CHO host cells has theamino acid sequence of amino acid residues 35 to 207 of FIG. 15 (SEQ IDNO:19) and amino acid residues 46 to 218 of FIG. 19 (SEQ ID NO:25).

Clone DNA114876 (designated DNA114876-2534) was deposited with ATCC andwas assigned ATCC deposit no. 203973. The full length hIL-1Ra1V proteinshown in FIG. 19 (SEQ ID NO:25) has an estimated molecular weight ofabout 24,124 and a pI of about 6.1.

Based on a sequence alignment analysis of the full length sequence (SEQID NO:25), hIL-1Ra1V shows significant amino acid sequence identity tohIL-1Raβ. hIL-1Ra1V is believed to be an allelic variant of hIL-1Ra1L.

Example 13 IL-18 Receptor and IL-1 Receptor Binding of hIL-1Ra1S

To facilitate the characterization of hIL-1Ra1S, a PCR fragment encodingamino acid residues 39-167 in the ORF of clone DNA102044 (FIG. 16; SEQID NO:21) was cloned into pCMV1FLAG (IBI Kodak, described in Pan et al.,Science, 276: 111-113) as an in-frame fusion to a NH₂-terminalpreprotrypsin leader sequence and FLAG tag encoded by the vector to formplasmid pCMV1FLAG-IL-1Ra1S. Plasmid pCMV1FLAG-IL18R-ECD-Fc was obtainedas described in Example 9 above.

Human embryonic kidney 293 cells were grown in high glucose DMEM(Genentech, Inc). The cells were seeded at 3-4×10⁶ per plate (100 mm)and co-transfected with pCMV1FLAG-hIL-1Ra1S and pCMV1FLAG-IL18R-ECD-Fcby means of calcium phosphate precipitation. The media were changed 12hours post transfection. The resultant conditioned media (10 ml each)were harvested after a further 70-74 hour incubation, clarified bycentrifugation, aliquoted and stored at −70° C. The receptor-Fc andligand complex from 1.5 ml conditioned medium was immunoprecipitatedwith protein G-Sepharose, washed three times with buffer containing 50mM Hepes, pH7.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and a proteaseinhibitor cocktail (BMB) and resolved on a 10-20% SDS-PAGE gel. Thebound ligand was identified by immunoblotting using anti-FLAG monoclonalantibody (BMB).

The immunoblotting results indicated that the secreted FLAGhIL-1Ra1Sfusion protein bound to IL-18R ECD. These data show that hIL-1Ra1S couldbe an agonist or antagonist of IL-18R.

Example 14 hIL-1Ra1V, hIL-1Ra1L and hIL-1Ra3 Processing

cDNAs encoding full-length hIL-1Ra1V (amino acids 1-218 in the ORF ofclone DNA114876 shown in FIG. 19 (SEQ ID NO:25)), full length hIL-1Ra1L(amino acids 1-207 in the ORF of clone DNA102043 shown in FIG. 15 (SEQID NO:19)), and full length hIL-1Ra3 (amino acids 1-155 in the ORF ofclone DNA96787 shown in FIG. 7 (SEQ ID NO:13)) were each cloned into apRK7 expression vector as an in-frame fusion with a carboxy-terminalFLAG-tag sequence. In preparation for mammalian cell transienttransfections, CHO DP12 cells were seeded at 4×10⁶ cells per plate (100mm petri dish) in growth medium (PS20, 5% FBS, 1×GHT, 1× pen/strep, 1×L-glutamine) the day before transfection. On the day of transfection,cells were washed with PBS and fed with 10 ml serum-free transfectionmedium (PS20, 1×GHT). DNA-lipid transfection mixtures were prepared byadding stepwise into eppendorf tubes (1) 400 μl transfection medium(PS20, 1×GHT); (2) 12 μg DNA; (3) 10 μg poly-lysine; and (4) 50 μlDosper liposomal transfection reagent (Boehringer Mannheim). TheDNA-lipid mixtures were incubated for 15 minutes at room temperature andthen added dropwise to cell culture plates. Cells were incubatedovernight at 37° C. On the day after transfection, cells were washedwith PBS, fed with 10 ml serum-free production medium (PS24, 10 mg/Linsulin, 1× trace elements, 1.4 mg/L lipid EtOH), and placed in a 32° C.incubator. After 5 days, the culture media containing the expressedproteins were harvested and cleared by centrifugation. For peptidesequencing of each expressed protein, 5-10 ml of the conditioned mediumcontaining the expressed protein was incubated with monoclonal anti-FLAGantibody (Boehringer Mannheim) coupled to agarose beads. Theimmunoprecipitated FLAG-tag proteins were extensively washed with 1%NP-40 buffer (125 mM NaCl, 1 mM EDTA and 50 mM Tris-HCl, pH 7.4). Theimmunoprecipitates were run on a SDS polyacrylamide gel, the separatedpolypeptides on the gel were transferred to a PVDF membrane, the PVDFmembrane was stained with Coomassie blue, and the corresponding proteinbands were excised from the membrane. The amino-terminal proteinsequences were obtained by conventional methods.

The processed N-terminal sequence of both of the hIL-1Ra1L and hIL-1Ra1Vpolypeptides was determined to be VHTSPKVKN (SEQ ID NO:31).Approximately 50% of hIL-1Ra1L and hIL-1Ra1V material recovered fromconditioned media exhibited the processed N-terminal sequence,indicating that the CHO host cells secreted a processed formcorresponding to amino acid residues 35 to 207 in the amino acidsequence of FIG. 15 (SEQ ID NO:19) and amino acid residues 46 to 218 inthe amino acid sequence of FIG. 19 (SEQ ID NO:25). The remaining 50% ofthe hIL-1Ra1L and hIL-1Ra1V material recovered from conditioned mediaexhibited an unprocessed N-terminus, indicating that the CHO host cellsalso secreted unprocessed forms of hIL-1Ra1L and hIL-1Ra1V correspondingto amino acid residues 1 to 207 in the amino acid sequence of FIG. 15(SEQ ID NO:19) and to amino acid residues 1 to 218 in the amino acidsequence of FIG. 19 (SEQ ID NO:25), respectively.

The processed N-terminal sequence of both of the hIL-1Ra3 and mIL-1Ra3polypeptides was determined to be VLSGALCFRM (SEQ ID NO:33).Approximately 100% of the hIL-1Ra3 and mIL-1Ra3 material recovered fromconditioned media exhibited the processed N-terminal sequence,indicating that the CHO host cells secreted processed forms of hIL-1Ra3and mIL-1Ra3 that lack the N-terminal methionine and correspond to aminoacid residues 2 to 155 in the amino acid sequence of FIG. 7 (SEQ IDNO:13) and amino acid residues 2 to 155 in the amino acid sequence ofFIG. 9 (SEQ ID NO:16), respectively.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,USA (ATCC): Material ATCC Dep. No. Deposit Date pSPORT1-based plasmid203586 Jan. 12, 1999 DNA92929-2534 pCMV-1Flag-pcmv5 plasmid 203587 Jan.12, 1999 DNA96786-2534 pT7T3D-Pac plasmid 203588 Jan. 12, 1999DNA85066-2534 pINCY-based plasmid 203589 Jan. 12, 1999 DNA96787-2534pT7T3D-Pac plasmid 203590 Jan. 12, 1999 DNA92505-2534 pRK7-based plasmid203846 Mar. 16, 1999 DNA102043-2534 pRK7-based plasmid 203855 Mar. 16,1999 DNA102044-2534 pRK7-based plasmid 203973 Apr. 27, 1999DNA114876-2534

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures of the deposits for30 years from the date of deposit. The deposits will be made availableby ATCC under the terms of the Budapest Treaty, and subject to anagreement between Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the cultures of the depositsto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 886OG 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the constructs deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.TABLE 2A PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein% amino acid sequence identity = (the number of identically matchingamino acid residues between the two polypeptide sequences as determinedby ALIGN-2) divided by (the total number of amino acid residues of thePRO polypeptide) = 5 divided by 15 = 33.3%

TABLE 2B PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein% amino acid sequence identity = (the number of identically matchingamino acid residues between the two polypeptide sequences as determinedby ALIGN-2) divided by (the total number of amino acid residues of thePRO polypeptide) = 5 divided by 10 = 50%

TABLE 2C PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) ComparisonNNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA% nucleic acid sequence identity = (the number of identically matchingnucleotides between the two nucleic acid sequences as determined byALIGN-2) divided by (the total number of nucleotides of the PRO-DNAnucleic acid sequence) = 6 divided by 14 = 42.9%

TABLE 2D PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNANNNNLLLVV (Length = 9 nucleotides)% nucleic acid sequence identity = (the number of identically matchingnucleotides between the two nucleic acid sequences as determined byALIGN-2) divided by (the total number of nucleotides of the PRO-DNAnucleic acid sequence) = 4 divided by 12 = 33.3%

TABLE 3A /*  *  * C—C increased from 12 to 15  * Z is average of EQ  * Bis average of ND  * match with stop is _M; stop-stop = 0; J (joker)match = 0  */ #define _M  −8  /* value of a match with a stop */ int_day[26][26] = { /*  A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/ /* A */  { 2, 0,−2, 0, 0,−4, 1,−1,−1, 0,−1,−2,−1, 0,_M, 1, 0,−2, 1,1, 0, 0,−6, 0,−3, 0}, /* B */  { 0, 3,−4, 3, 2,−5, 0, 1,−2, 0, 0,−3,−2,2,_M,−1, 1, 0, 0, 0, 0,−2,−5, 0,−3, 1}, /* C */ {−2,−4,15,−5,−5,−4,−3,−3,−2, 0,−5,−6,−5,−4,_M,−3,−5,−4, 0,−2, 0,−2,−8,0, 0,−5}, /* D */  { 0, 3,−5, 4, 3,−6, 1, 1,−2, 0, 0,−4,−3, 2,_M,−1,2,−1, 0, 0, 0,−2,−7, 0,−4, 2}, /* E */  { 0, 2,−5, 3, 4,−5, 0, 1,−2, 0,0,−3,−2, 1,_M,−1, 2,−1, 0, 0, 0,−2,−7, 0,−4, 3}, /* F */ {−4,−5,−4,−6,−5, 9,−5,−2, 1, 0,−5, 2, 0,−4,_M,−5,−5,−4,−3,−3, 0,−1, 0,0, 7,−5}, /* G */  { 1, 0,−3, 1, 0,−5, 5,−2,−3, 0,−2,−4,−3,0,_M,−1,−1,−3, 1, 0, 0,−1,−7, 0,−5, 0}, /* H */  {−1, 1,−3, 1, 1,−2,−2,6,−2, 0, 0,−2,−2, 2,_M, 0, 3, 2,−1,−1, 0,−2,−3, 0, 0, 2}, /* I */ {−1,−2,−2,−2,−2, 1,−3,−2, 5, 0,−2, 2, 2,−2,_M,−2,−2,−2,−1, 0, 0, 4,−5,0,−1,−2}, /* J */  { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0}, /* K */  {−1, 0,−5, 0, 0,−5,−2, 0,−2, 0,5,−3, 0, 1,_M,−1, 1, 3, 0, 0, 0,−2,−3, 0,−4, 0}, /* L */ {−2,−3,−6,−4,−3, 2,−4,−2, 2, 0,−3, 6, 4,−3,_M,−3,−2,−3,−3,−1, 0, 2,−2,0,−1,−2}, /* M */  {−1,−2,−5,−3,−2, 0,−3,−2, 2, 0, 0, 4, 6,−2,_M,−2,−1,0,−2,−1, 0, 2,−4, 0,−2,−1}, /* N */  { 0, 2,−4, 2, 1,−4, 0, 2,−2, 0,1,−3,−2, 2,_M,−1, 1, 0, 1, 0, 0,−2,−4, 0,−2, 1}, /* O */ {_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M}, /* P */  { 1,−1,−3,−1,−1,−5,−1,0,−2, 0,−1,−3,−2,−1,_M, 6, 0, 0, 1, 0, 0,−1,−6, 0,−5, 0}, /* Q */  { 0,1,−5, 2, 2,−5,−1, 3,−2, 0, 1,−2,−1, 1,_M, 0, 4, 1,−1,−1, 0,−2,−5, 0,−4,3}, /* R */  {−2, 0,−4,−1,−1,−4,−3, 2,−2, 0, 3,−3, 0, 0,_M, 0, 1, 6,0,−1, 0,−2, 2, 0,−4, 0}, /* S */  { 1, 0, 0, 0, 0,−3, 1,−1,−1, 0,0,−3,−2, 1,_M, 1,−1, 0, 2, 1, 0,−1,−2, 0,−3, 0}, /* T */  { 1, 0,−2, 0,0,−3, 0,−1, 0, 0, 0,−1,−1, 0,_M, 0,−1,−1, 1, 3, 0, 0,−5, 0,−3, 0}, /* U*/  { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, 0}, /* V */  { 0,−2,−2,−2,−2,−1,−1,−2, 4, 0,−2, 2,2,−2,_M,−1,−2,−2,−1, 0, 0, 4,−6, 0,−2,−2}, /* W */  {−6,−5,−8,−7,−7,0,−7,−3,−5, 0,−3,−2,−4,−4,_M,−6,−5, 2,−2,−5, 0,−6,17, 0, 0,−6}, /* X */ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0}, /* Y */  {−3,−3, 0,−4,−4, 7,−5, 0,−1,0,−4,−1,−2,−2,_M,−5,−4,−4,−3,−3, 0,−2, 0, 0,10,−4}, /* Z */  { 0, 1,−5,2, 3,−5, 0, 2,−2, 0, 0,−2,−1, 1,_M, 0, 3, 0, 0, 0, 0,−2,−6, 0,−4, 4} };

TABLE 3B /*  */ #include <stdio.h> #include <ctype.h> #define MAXJMP 16/* max jumps in a diag */ #define MAXGAP 24 /* don't continue topenalize gaps larger than this */ #define JMPS 1024 /* max jmps in anpath */ #define MX 4 /* save if there's at least MX-1 bases since lastjmp */ #define DMAT 3 /* value of matching bases */ #define DMIS 0 /*penalty for mismatched bases */ #define DINS0 8 /* penalty for a gap */#define DINS1 1 /* penalty per base */ #define PINS0 8 /* penalty for agap */ #define PINS1 4 /* penalty per residue */ struct jmp { shortn[MAXJMP]; /* size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /*base no. of jmp in seq x */ }; /* limits seq to 2{circumflex over ( )}16−1 */ struct diag { int score; /* score at last jmp */ long offset; /*offset of prev block */ short ijmp; /* current jmp index */ struct jmpjp; /* list of jmps */ }; struct path { int spc; /* number of leadingspaces */ short n[JMPS];/* size of jmp (gap) */ int x[JMPS];/* loc ofjmp (last elem before gap) */ }; char *ofile; /* output file name */char *namex[2]; /* seq names: getseqs( ) */ char *prog; /* prog name forerr msgs */ char *seqx[2]; /* seqs: getseqs( ) */ int dmax; /* bestdiag: nw( ) */ int dmax0; /* final diag */ int dna; /* set if dna: main() */ int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /*total gaps in seqs */ int len0, len1; /* seq lens */ int ngapx, ngapy;/* total size of gaps */ int smax; /* max score: nw( ) */ int *xbm; /*bitmap for matching */ long offset; /* current offset in jmp file */struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds pathfor seqs */ char *calloc( ), *malloc( ), *index( ), *strcpy( ); char*getseq( ), *g_calloc( );

TABLE 3C /* Needleman-Wunsch alignment program  *  * usage: progs file1file2  * where file1 and file2 are two dna or two protein sequences. * The sequences can be in upper- or lower-case an may contain ambiguity * Any lines beginning with ‘;’, ‘>’ or ‘<’ are ignored  * Max filelength is 65535 (limited by unsigned short x in the jmp struct)  * Asequence with ⅓ or more of its elements ACGTU is assumed to be DNA * Output is in the file “align.out”  *  * The program may create a tmpfile in /tmp to hold info about traceback.  * Original version developedunder BSD 4.3 on a vax 8650  */ #include “nw.h” #include “day.h” static_dbval[26] = { 1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0}; static _pbval[26] = { 1, 2|(1 < < (‘D’−‘A’))|(1 < < (‘N’−‘A’)), 4, 8,16, 32, 64, 128, 256, 0xFFFFFFF, 1 < < 10, 1 < < 11, 1 < < 12, 1 < < 13,1 < < 14, 1 < < 15, 1 < < 16, 1 < < 17, 1 < < 18, 1 < < 19, 1 < < 20, 1< < 21, 1 < < 22, 1 < < 23, 1 < < 24, 1 < < 25|(1 < < (‘E’−‘A’))|(1 < <(‘Q’−‘A’)) }; main main(ac, av) int ac; char *av[ ]; { prog = av[0]; if(ac != 3) { fprintf(stderr,“usage: %s file1 file2\n”, prog);fprintf(stderr,“where file1 and file2 are two dna or two proteinsequences.\n”); fprintf(stderr,“The sequences can be in upper- orlower-case\n”); fprintf(stderr,“Any lines beginning with ‘;’ or ‘<’ areignored\n”); fprintf(stderr,“Output is in the file \“align.out\”\n”);exit(1); } namex[0] = av[1]; namex[1] = av[2]; seqx[0] =getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1); xbm = (dna)?_dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ ofile =“align.out”; /* output file */ nw( ); /* fill in the matrix, get thepossible jmps */ readjmps( ); /* get the actual jmps */ print( ); /*print stats, alignment */ cleanup(0); /* unlink any tmp files */ }

TABLE 3D /* do the alignment, return best score: main( )  * dna: valuesin Fitch and Smith, PNAS, 80, 1382-1386, 1983  * pro: PAM 250 values  *When scores are equal, we prefer mismatches to any gap, prefer  * a newgap to extending an ongoing gap, and prefer a gap in seqx  * to a gap inseq y.  */ nw( ) nw { char *px, *py; /* seqs and ptrs */ int *ndely,*dely; /* keep track of dely */ int ndelx, delx; /* keep track of delx*/ int *tmp; /* for swapping row0, row1 */ int mis; /* score for eachtype */ int ins0, ins1; /* insertion penalties */ register id; /*diagonal index */ register ij; /* jmp index */ register *col0, *col1; /*score for curr, last row */ register xx, yy; /* index into seqs */ dx =(struct diag *)g_calloc(“to get diags”, len0+len1+1, sizeof(structdiag)); ndely = (int *)g_calloc(“to get ndely”, len1+1, sizeof(int));dely = (int *)g_calloc(“to get dely”, len1+1, sizeof(int)); col0 = (int*)g_calloc(“to get col0”, len1+1, sizeof(int)); col1 = (int*)g_calloc(“to get col1”, len1+1, sizeof(int)); ins0 = (dna)? DINS0 :PINS0; ins1 = (dna)? DINS1 : PINS1; smax = −10000; if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1; yy <= len1; yy++) { col0[yy] =dely[yy] = col0[yy−1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */ } else for (yy = 1; yy <= len1; yy++) dely[yy] =−ins0; /* fill in match matrix  */ for (px = seqx[0], xx = 1; xx <=len0; px++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx == 1) col1[0] = delx = −(ins0+ins1); else col1[0] = delx =col0[0] − ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx =0; }

TABLE 3E ...nw for (py = seqx[1], yy = 1; yy <= len1; py++, yy++) {  mis = col0[yy−1];   if (dna)     mis += (xbm[*px−‘A’]&xbm[*py−‘A’])?DMAT : DMIS;   else     mis += _day[*px−‘A’][*py−‘A’];   /* updatepenalty for del in x seq;    * favor new del over ongong del    * ignoreMAXGAP if weighting endgaps    */   if (endgaps || ndely[yy] < MAXGAP) {    if (col0[yy] − ins0 >= dely[yy]) {       dely[yy] = col0[yy] −(ins0+ins1);       ndely[yy] = 1;     } else {       dely[yy] −= ins1;      ndely[yy]++;     }   } else {     if (col0[yy] − (ins0+ins1) >=dely[yy]) {       dely[yy] = col0[yy] − (ins0+ins1);       ndely[yy] =1;     } else       ndely[yy]++;   }   /* update penalty for del in yseq;    * favor new del over ongong del    */   if (endgaps || ndelx <MAXGAP) {     if (col1[yy−1] − ins0 >= delx) {       delx = col1[yy−1] −(ins0+ins1);       ndelx = 1;     } else {       delx −= ins1;      ndelx++;     }   } else {     if (col1[yy−1] − (ins0+ins1) >=delx) {       delx = col1[yy−1] − (ins0+ins1);       ndelx = 1;     }else       ndelx++;   }   /* pick the maximum score; we're favoring    *mis over any del and delx over dely    */

TABLE 3F ..nw id = xx − yy + len1 − 1; if (mis >= delx && mis >=dely[yy])   col1[yy] = mis; else if (delx >= dely[yy]) {   col1[yy] =delx;   ij = dx[id].ijmp;   if (dx[id].jp.n[0] && (!dna || (ndelx >=MAXJMP   && xx > dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) {    dx[id].ijmp++;     if (++ij >= MAXJMP) {             writejmps(id);            ij = dx[id].ijmp = 0;             dx[id].offset = offset;            offset += sizeof(struct jmp) + sizeof(offset);           }        }         dx[id].jp.n[ij] = ndelx;         dx[id].jp.x[ij] = xx;        dx[id].score = delx;       }       else {         col1[yy] =dely[yy];         ij = dx[id].ijmp;   if (dx[id].jp.n[0] && (!dna ||(ndely[yy] >= MAXJMP         && xx > dx[id].jp.x[ij]+MX) ||        mis > dx[id].score+DINS0)) {           dx[id].ijmp++;          if (++ij >= MAXJMP) {             writejmps(id);            ij = dx[id].ijmp = 0;             dx[id].offset = offset;            offset += sizeof(struct jmp) + sizeof(offset);           }        }         dx[id].jp.n[ij] = −ndely[yy];         dx[id].jp.x[ij]= xx;         dx[id].score = dely[yy];       }       if (xx == len0 &&yy < len1) {         /* last col          */         if (endgaps)          col1[yy] − = ins0+ins1*(len1−yy);         if (col1[yy] > smax){           smax = col1[yy];           dmax = id;         }       }    }     if (endgaps && xx < len0)       col1[yy−1] −=ins0+ins1*(len0−xx);     if (col1[yy−1] > smax) {       smax =col1[yy−1];       dmax = id;     }     tmp = col0; col0 = col1; col1 =tmp;   }   (void) free((char *)ndely);   (void) free((char *)dely);  (void) free((char *)col0);   (void) free((char *)col1); }

TABLE 3G /*  *  * print( ) -- only routine visible outside this module *  * static:  * getmat( ) -- trace back best path, count matches:print( )  * pr_align( ) -- print alignment of described in array p[ ]:print( )  * dumpblock( ) -- dump a block of lines with numbers, stars:pr_align( )  * nums( ) -- put out a number line: dumpblock( )  *putline( ) -- put out a line (name, [num], seq, [num]): dumpblock( )  *stars( ) - - put a line of stars: dumpblock( )  * stripname( ) -- stripany path and prefix from a seqname  */ #include “nw.h” #define SPC 3#define P_LINE 256 /* maximum output line */ #define P_SPC 3 /* spacebetween name or num and seq */ extern _day[26][26]; int olen; /* setoutput line length */ FILE *fx; /* output file */ print( ) print { intlx, ly, firstgap, lastgap;  /* overlap */ if ((fx = fopen(ofile, “w”))== 0) { fprintf(stderr,“%s: can't write %s\n”, prog, ofile); cleanup(1);} fprintf(fx, “<first sequence: %s (length = %d)\n”., namex[0], len0);fprintf(fx, “<second sequence: %s (length = %d)\n”, namex[1], len1);olen = 60; lx = len0; ly = len1; firstgap = lastgap = 0; if (dmax < len1− 1) {  /* leading gap in x */ pp[0].spc = firstgap = len1 − dmax − 1;ly −= pp[0].spc; } else if (dmax > len1 − 1) { /* leading gap in y */pp[1].spc = firstgap = dmax − (len1 − 1); lx −= pp[1].spc; } if (dmax0 <len0 − 1) {  /* trailing gap in x */ lastgap = len0 − dmax0 −1; lx −=lastgap; } else if (dmax0 > len0 − 1) { /* trailing gap in y */ lastgap= dmax0 − (len0 − 1); ly −= lastgap; } getmat(lx, ly, firstgap,lastgap); pr_align( ); }

TABLE 3H /*  * trace back the best path, count matches  */ staticgetmat(lx, ly, firstgap, lastgap) getmat int lx, ly; /* “core” (minusendgaps) */ int firstgap, lastgap; /* leading trailing overlap */ { intnm, i0, i1, siz0, siz1; char outx[32]; double pct; register n0, n1;register char *p0, *p1; /* get total matches, score  */ i0 = i1 = siz0 =siz1 = 0; p0 = seqx[0] + pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 =pp[1].spc + 1; n1 = pp[0].spc + 1; nm = 0; while ( *p0 && *p1 ) { if(siz0) { p1++; n1++; siz0−−; } else if (siz1) { p0++; n0++; siz1−−; }else { if (xbm[*p0−‘A’]&xbm[*p1−‘A’])   nm++; if (n0++ == pp[0].x[i0])  siz0 = pp[0].n[i0++]; if (n1++ == pp[1].x[i1])   siz1 = pp[1].n[i1++];p0++; p1++; } } /* pct homology:  * if penalizing endgaps, base is theshorter seq  * else, knock off overhangs and take shorter core  */ if(endgaps) lx = (len0 < len1)? len0 : len1; else lx = (lx < ly)? lx : ly;pct = 100.*(double)nm/(double)lx; fprintf(fx, “\n”); fprintf(fx, “< %dmatch%s in an overlap of %d: %.2f percent similarity\n”, nm, (nm == 1)?“” : “es”, lx, pct);

TABLE 3I fprintf(fx, “<gaps in first sequence: %d”, gapx); ...getmat if(gapx) { (void) sprintf(outx, “ (%d %s%s)”, ngapx, (dna)?“base”:“residue”, (ngapx == 1)? “”:“s”); fprintf(fx,“%s”, outx);fprintf(fx, “, gaps in second sequence: %d”, gapy); if (gapy) { (void)sprintf(outx, “ (%d %s%s)”, ngapy, (dna)? “base”:“residue”, (ngapy ==1)? “”:“s”); fprintf(fx,“%s”, outx); } if (dna) fprintf(fx, “\n<score:%d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n”, smax,DMAT, DMIS, DINS0, DINS1); else fprintf(fx, “\n<score: %d (Dayhoff PAM250 matrix, gap penalty = %d + %d per residue)\n”, smax, PINS0, PINS1);if (endgaps) fprintf(fx, “<endgaps penalized. left endgap: %d %s%s,right endgap: %d %s%s\n”, firstgap, (dna)? “base” : “residue”, (firstgap== 1)? “” : “s”, lastgap, (dna)? “base” : “residue”, (lastgap == 1)? “”: “s”); else fprintf(fx, “<endgaps not penalized\n”); } static nm; /*matches in core -- for checking */ static lmax; /* lengths of strippedfile names */ static ij[2]; /* jmp index for a path */ static nc[2]; /*number at start of current line */ static ni[2]; /* current elem number-- for gapping */ static siz[2]; static char *ps[2]; /* ptr to currentelement */ static char *po[2]; /* ptr to next output char slot */ staticchar out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* setby stars( ) */ /*  * print alignment of described in struct path pp[ ] */ static pr_align( ) pr_align { int nn; /* char count */ int more;register i; for (i = 0, lmax = 0; i < 2; i++) { nn =stripname(namex[i]); if (nn > lmax) lmax = nn; nc[i] = 1; ni[i] = 1;siz[i] = ij[i] = 0; ps[i] = seqx[i]; po[i] = out[i];

TABLE 3J  for (nn = nm = 0, more = 1; more; ) { ...pr_align   for (i =more = 0; i < 2; i++) {    /*     * do we have more of this sequence?   */    if (!*ps[i])     continue;    more++;    if (pp[i].spc) { /*leading space */     *po[i]++ = ‘ ’;     pp[i].spc−−;    }    else if(siz[i]) { /* in a gap */     *po[i]++ = ‘-’;     siz[i]−−;    }    else{ /* we're putting a seq element  */     *po[i] = *ps[i];     if(islower(*ps[i])) *ps[i] = toupper(*ps[i]);     po[i]++;     ps[i]++;    /*      * are we at next gap for this seq?      */     if (ni[i] ==pp[i].x[ij[i]]) { /*  * we need to merge all gaps  * at this location */ siz[i] = pp[i].n[ij[i]++]; while (ni[i] == pp[i].x[ij[i]])  siz[i]+= pp[i].n[ij[i]++];     }     ni[i]++;    }   }   if (++nn == olen ||!more && nn) {    dumpblock( );    for (i = 0; i < 2; i++)     po[i] =out[i];    nn = 0;   }  } } /*  * dump a block of lines, includingnumbers, stars: pr_align( )  */ static dumpblock( ) dumpblock { register i;  for (i = 0; i < 2; i++)   *po[i]−− = ‘\0’;

TABLE 3K ...dumpblock (void) putc(‘\n’, fx); for(i = 0; i < 2; i++) { if(*out[i] && (*out[i] != ‘ ’ || *(po[i]) != ‘ ’)) { if (i == 0) nums(i);if (i == 0 && *out[1]) stars( ); putline(i); if (i == 0 && *out[1])fprintf(fx, star); if (i == 1) nums(i); } } } /*  * put out a numberline: dumpblock( )  */ static nums(ix) nums int ix; /* index in out[ ]holding seq line */ { char nline[P_LINE]; register i, j; register char*pn, *px, *py; for (pn = nline, i = 0; i < lmax+P_SPC; i++, pn++) *pn =‘ ’; for (i = nc[ix], py = out[ix]; *py; py++, pn++) { if (*py == ‘ ’ ||*py == ‘-’) *pn = ‘ ’; else { if (i%10 == 0 || (i == 1 && nc[ix] != 1)){ j = (i < 0)? −i : i; for (px = pn; j; j /= 10, px−−) *px = j%10 + ‘0’;if (i < 0) *px = ‘-’; } else *pn = ‘ ’; i++; } } *pn = ‘\0’; nc[ix] = i;for (pn = nline; *pn; pn++) (void) putc(*pn, fx); (void) putc(‘\n’, fx);} /*  * put out a line (name, [num], seq, [num]): dumpblock( )  */static putline(ix) putline int ix; {

TABLE 3L ...putline int i; register char *px; for (px = namex[ix], i =0; *px && *px != ‘:’; px++, i++) (void) putc(*px, fx); for (; i <lmax+P_SPC; i++) (void) putc(‘ ’, fx); /* these count from 1:  * ni[ ]is current element (from 1)  * nc[ ] is number at start of current line */ for (px = out[ix]; *px; px++) (void) putc(*px&0x7F, fx); (void)putc(‘\n’, fx); } /*  * put a line of stars (seqs always in out[0],out[1]): dumpblock( )  */ static stars( ) stars { int i; register char*p0, *p1, cx, *px; if (!*out[0] || (*out[0] == ‘ ’ && *(po[0]) == ‘ ’)||   !*out[1] || (*out[1] == ‘ ’ && *(po[1]) == ‘ ’)) return; px = star;for (i = lmax+P_SPC; i; i−−) *px++ = ‘ ’; for (p0 = out[0], p1 = out[1];*p0 && *p1; p0++, p1++) { if (isalpha(*p0) && isalpha(*p1)) { if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) { cx = ‘*’; nm++; } else if (!dna &&_day[*p0−‘A’][*p1−‘A’] > 0) cx = ‘.’; else cx = ‘ ’; } else cx = ‘ ’;*px++ = cx; } *px++ = ‘\n’; *px = ‘\0’; }

TABLE 3M /*  * strip path or prefix from pn, return len: pr_align( )  */static stripname(pn) stripname    char *pn; /* file name (may be path)*/ {    register char *px, *py;    py = 0;    for (px = pn; *px; px++)if(*px == ‘/’) py = px + 1;    if (py) (void) strcpy(pn, py);   return(strlen(pn)); }

TABLE 3N /*  * cleanup( ) -- cleanup any tmp file  * getseq( ) -- readin seq, set dna, len, maxlen  * g_calloc( ) -- calloc( ) with errorcheckin  * readjmps( ) -- get the good jmps, from tmp file if necessary * writejmps( ) -- write a filled array of jmps to a tmp file: nw( )  */#include “nw.h” #include <sys/file.h> char *jname = “/tmp/homgXXXXXX”;/* tmp file for jmps */ FILE *fj; int cleanup( ); /* cleanup tmp file */long lseek( ); /*  * remove any tmp file if we blow  */ cleanup(i)cleanup int i; { if(fj) (void) unlink(jname); exit(i); } /*  * read,return ptr to seq, set dna, len, maxlen  * skip lines starting with ‘;’,‘<’, or ‘>’  * seq in upper or lower case */ char * getseq(file, len)getseq char *file; /* file name */ int *len; /* seq len */ { charline[1024], *pseq; register char *px, *py; int natgc, tlen; FILE *fp; if((fp = fopen(file,“r”)) == 0) { fprintf(stderr, “%s: can't read %s\n”,prog, file); exit(1); } tlen = natgc = 0; while (fgets(line, 1024, fp)){ if (*line == ‘;’ || *line == ‘<’ || *line == ‘>’) continue; for (px =line; *px != ‘\n’; px++) if (isupper(*px) || islower(*px)) tlen++; } if((pseq = malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr, “%s:malloc( ) failed to get %d bytes for %s\n”, prog, tlen+6, file);exit(1); } pseq[0] = pseq[1] = pseq[2] = pseq[3] = ‘\0’;

TABLE 3O ...getseq py = pseq + 4; *len = tlen; rewind(fp); while(fgets(line, 1024, fp)) { if (*line == ‘;’ || *line == ‘<’ || *line ==‘>’) continue; for (px = line; *px != ‘\n’; px++) { if (isupper(*px))*py++ = *px; else if (islower(*px)) *py++ = toupper(*px); if(index(“ATGCU”,*(py−1))) natgc++; } } *py++ = ‘\0’; *py = ‘\0’; (void)fclose(fp); dna = natgc > (tlen/3); return(pseq+4); } char *g_calloc(msg, nx, sz) g_calloc char *msg; /* program, calling routine */int nx, sz; /* number and size of elements */ { char *px, *calloc( ); if((px = calloc((unsigned)nx, (unsigned)sz)) = = 0) { if (*msg) {fprintf(stderr, “%s: g_calloc( ) failed %s (n = %d, sz = %d, prog, msg,nx, sz); exit(1); } } return(px); } /*  * get final jmps from dx[ ] ortmp file, set pp[ ], reset dmax: main( )  */ readjmps( ) readjmps { intfd = −1; int siz, i0, i1; register i, j, xx; if (fj) { (void)fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) { fprintf(stderr,“%s: can't open( ) %s\n”, prog, jname); cleanup(1); } } for (i = i0 = i1= 0, dmax0 = dmax, xx = len0; ; i++) { while (1) { for (j =dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j−−) ;

TABLE 3P ...readjmps    if (j < 0 && dx[dmax].offset && fj) {     (void)lseek(fd, dx[dmax].offset, 0);     (void) read(fd, (char *)&dx[dmax].jp,sizeof(struct jmp));     (void) read(fd, (char *)&dx[dmax].offset,    sizeof(dx[dmax].offset));     dx[dmax].ijmp = MAXJMP-1;    }    else    break;   }   if (i >= JMPS) {    fprintf(stderr, “%s: too many gapsin alignment\n”, prog);    cleanup(1);   }   if (j >= 0) {    siz =dx[dmax].jp.n[j];    xx = dx[dmax].jp.x[j];    dmax += siz;    if (siz <0) {       /* gap in second seq */     pp[1].n[i1] = −siz;     xx +=siz;     /* id = xx − yy + len1 − 1      */     pp[1].x[i1] = xx −dmax + len1 − 1;     gapy++;     ngapy −= siz; /* ignore MAXGAP whendoing endgaps */     siz = (−siz < MAXGAP || endgaps)? −siz : MAXGAP;    i1++;    }    else if (siz > 0) { /* gap in first seq */    pp[0].n[i0] = siz;     pp[0].x[i0] = xx;     gapx++;     ngapx +=siz; /* ignore MAXGAP when doing endgaps */     siz = (siz < MAXGAP ||endgaps)? siz : MAXGAP;     i0++;    }   }   else    break;  }  /*reverse the order of jmps   */  for (j = 0, i0−−; j < i0; j++, i0−−) {  i = pp[0].n[j]; pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i;   i =pp[0].x[j]; pp[0].x[j] = pp[0].x[i0]; pp[0].x[i0] = i;  }  for (j = 0,i1−−; j < i1; j++, i1−−) {   i = pp[1].n[j]; pp[1].n[j] = pp[1].n[i1];pp[1].n[i1] = i;   i = pp[1].x[j]; pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1]= i;  }  if (fd >= 0)   (void) close(fd);  if (fj) {   (void)unlink(jname);   fj = 0;   offset = 0;

TABLE 3Q /* * write a filled jmp struct offset of the prev one (if any):nw( )  */ writejmps(ix) writejmps   int ix; {   char *mktemp( );   if(!fj) { if (mktemp(jname) < 0) {    fprintf(stderr, “%s: can't mktemp( )%s\n”, prog,    jname);    cleanup(1); } if ((fj = fopen(jname, “w”)) ==0) {    fprintf(stderr, “%s: can't write %s\n”, prog, jname);   exit(1); }   }   (void) fwrite((char *)&dx[ix].jp, sizeof(structjmp), 1, fj);   (void) fwrite((char *)&dx[ix].offset,sizeof(dx[ix].offset), 1, fj); }

1. An isolated DNA molecule selected from the group consisting of: (1) aDNA molecule encoding an hIL-1Ra1 polypeptide comprising the amino acidsequence of amino acid residues from about 37 to about 203 of FIG. 2(SEQ ID NO:5); (2) a DNA molecule encoding an hIL-1Ra1 polypeptidecomprising the amino acid sequence of amino acid residues from about 15to about 193 of FIG. 3 (SEQ ID NO:7); (3) a DNA molecule encoding anhIL-1Ra2 polypeptide comprising the amino acid sequence of amino acidresidues from about 1 to about 134 of FIG. 5 (SEQ ID NO:10); (4) a DNAmolecule encoding an hIL-1Ra3 polypeptide comprising the amino acidsequence of amino acid residues from about 95 to about 134 of FIG. 7(SEQ ID NO:13); (5) a DNA molecule encoding a mIL-1Ra3 polypeptidecomprising the amino acid sequence of amino acid residues from about 95to about 134 of FIG. 9 (SEQ ID NO:16); (6) a DNA molecule encoding anhIL-1Ra1L polypeptide comprising the amino acid sequence of amino acidresidues from about 26 to about 207 of FIG. 15 (SEQ ID NO:19); (7) a DNAmolecule encoding an hIL-1Ra1S polypeptide comprising the amino acidsequence of amino acid residues from about 26 to about 167 of FIG. 16(SEQ ID NO:21); (8) a DNA molecule encoding an hIL-1Ra1V polypeptidecomprising the amino acid sequence of amino acid residues from about 46to about 218 of FIG. 19 (SEQ ID NO:25); and (9) the complement of any ofthe DNA molecules of (1)-(8).
 2. The isolated DNA molecule of claim 1selected from the group consisting of: (1) a DNA molecule encoding anhIL-1Ra1 polypeptide comprising the amino acid sequence of amino acidresidues from about 37 to about 203 of FIG. 2 (SEQ ID NO:5); (2) a DNAmolecule encoding an hIL-1Ra1 polypeptide comprising the amino acidsequence of amino acid residues from about 15 to about 193 of FIG. 3(SEQ ID NO:7); (3) a DNA molecule encoding an hIL-1Ra2 polypeptidecomprising the amino acid sequence of amino acid residues from about 1to about 134 of FIG. 5 (SEQ ID NO:10); (4) a DNA molecule encoding anhIL-1Ra3 polypeptide comprising the amino acid sequence of amino acidresidues from about 95 to about 134 of FIG. 7 (SEQ ID NO:13); (5) a DNAmolecule encoding a mIL-1Ra3 polypeptide comprising the amino acidsequence of amino acid residues from about 95 to about 134 of FIG. 9(SEQ ID NO:16); and (6) the complement of any of the DNA molecules of(1)-(5).
 3. The isolated DNA molecule of claim 1 selected from the groupconsisting of: (1) a DNA molecule encoding an hIL-1Ra1L polypeptidecomprising the amino acid sequence of amino acid residues from about 26to about 207 of FIG. 15 (SEQ ID NO:19); (2) a DNA molecule encoding anhIL-1Ra1S polypeptide comprising the amino acid sequence of amino acidresidues from about 26 to about 167 of FIG. 16 (SEQ ID NO:21); (3) a DNAmolecule encoding an hIL-1Ra1V polypeptide comprising the amino acidsequence of amino acid residues from about 46 to about 218 of FIG. 19(SEQ ID NO:25); and (4) the complement of any of the DNA molecules of(1)-(3).
 4. The isolated DNA molecule of claim 3 selected from the groupconsisting of: (1) a DNA molecule encoding an hIL-1Ra1L polypeptidecomprising the amino acid sequence of amino acid residues from about 1to about 207 of FIG. 15 (SEQ ID NO:19); (2) a DNA molecule encoding anhIL-1Ra1S polypeptide comprising the amino acid sequence of amino acidresidues from about 1 to about 167 of FIG. 16 (SEQ ID NO:21); (3) a DNAmolecule encoding an hIL-1Ra1V polypeptide comprising the amino acidsequence of amino acid residues from about 1 to about 218 of FIG. 19(SEQ ID NO:25); and (4) the complement of any of the DNA molecules of(1)-(2).
 5. The isolated DNA molecule of claim 2 selected from the groupconsisting of: (1) a DNA molecule encoding an hIL-1Ra3 polypeptidecomprising the amino acid sequence of amino acid residues from about 34to about 155 of FIG. 7 (SEQ ID NO:13); (2) a DNA molecule encoding amIL-1Ra3 polypeptide comprising the amino acid sequence of amino acidresidues from about 34 to about 155 of FIG. 9 (SEQ ID NO:16); and (3)the complement of any of the DNA molecules of (1)-(2).
 6. The isolatedDNA molecule of claim 5 selected from the group consisting of: (1) a DNAmolecule encoding an hIL-1Ra3 polypeptide comprising the amino acidsequence of amino acid residues from about 2 to about 155 of FIG. 7 (SEQID NO:13); (2) a DNA molecule encoding a mIL-1Ra3 polypeptide comprisingthe amino acid sequence of amino acid residues from about 2 to about 155of FIG. 9 (SEQ ID NO:16); and (3) the complement of any of the DNAmolecules of (1)-(2).
 7. The isolated DNA molecule of claim 1 selectedfrom the group consisting of: (1) a DNA molecule which encodes anhIL-1Ra1 polypeptide, and which comprises the nucleic acid sequence ofnucleotide positions from about 118 to about 618 in the sense strand ofFIG. 2 (SEQ ID NO:4); (2) a DNA molecule which encodes an hIL-1Ra1polypeptide, and which comprises the nucleic acid sequence of nucleotidepositions from about 145 to about 681 in the sense strand of FIG. 3 (SEQID NO:6); (3) a DNA molecule which encodes an hIL-1Ra2 polypeptide, andwhich comprises the nucleic acid sequence of nucleotide positions fromabout 96 to about 497 in the sense strand of FIG. 5 (SEQ ID NO:9); (4) aDNA molecule which encodes an hIL-1Ra3 polypeptide, and which comprisesthe nucleic acid sequence of nucleotide positions from about 283 toabout 402 in the sense strand of FIG. 7 (SEQ ID NO:12); (5) a DNAmolecule which encodes a mIL-1Ra3 polypeptide, and which comprises thenucleic acid sequence of nucleotide positions from about 427 to about546 in the sense strand of FIG. 9 (SEQ ID NO:15); (6) a DNA moleculewhich encodes an hIL-1Ra1L polypeptide, and which comprises the nucleicacid sequence of nucleotide positions from about 79 to about 624 in thesense strand of FIG. 15 (SEQ ID NO:18); (7) a DNA molecule which encodesan hIL-1Ra1S polypeptide, and which comprises the nucleic acid sequenceof nucleotide positions from about 79 to about 504 in the sense strandof FIG. 16 (SEQ ID NO:20); (8) a DNA molecule which encodes an hIL-1Ra1Vpolypeptide, and which comprises the nucleic acid sequence of nucleotidepositions from about 208 to about 726 in the sense strand of FIG. 19(SEQ ID NO:24); and (9) the complement of any of the DNA molecules of(1)-(8).
 8. The isolated DNA molecule of claim 7 selected from the groupconsisting of: (1) a DNA molecule which encodes an hIL-1Ra1 polypeptide,and which comprises the nucleic acid sequence of nucleotide positionsfrom about 118 to about 618 in the sense strand of FIG. 2 (SEQ ID NO:4);(2) a DNA molecule which encodes an hIL-1Ra1 polypeptide, and whichcomprises the nucleic acid sequence of nucleotide positions from about145 to about 681 in the sense strand of FIG. 3 (SEQ ID NO:6); (3) a DNAmolecule which encodes an hIL-1Ra2 polypeptide, and which comprises thenucleic acid sequence of nucleotide positions from about 96 to about 497in the sense strand of FIG. 5 (SEQ ID NO:9); (4) a DNA molecule whichencodes an hIL-1Ra3 polypeptide, and which comprises the nucleic acidsequence of nucleotide positions from about 283 to about 402 in thesense strand of FIG. 7 (SEQ ID NO:12); (5) a DNA molecule which encodesa mIL-1Ra3 polypeptide, and which comprises the nucleic acid sequence ofnucleotide positions from about 427 to about 546 in the sense strand ofFIG. 9 (SEQ ID NO:15); and (6) the complement of any of the DNAmolecules of (1)-(5).
 9. The isolated DNA molecule of claim 7 selectedfrom the group consisting of: (1) a DNA molecule which encodes anhIL-1Ra1L polypeptide, and which comprises the nucleic acid sequence ofnucleotide positions from about 79 to about 624 in the sense strand ofFIG. 15 (SEQ ID NO:18); (2) a DNA molecule which encodes an hIL-1Ra1Spolypeptide, and which comprises the nucleic acid sequence of nucleotidepositions from about 79 to about 504 in the sense strand of FIG. 16 (SEQID NO:20); (3) a DNA molecule which encodes an hIL-1Ra1V polypeptide,and which comprises the nucleic acid sequence of nucleotide positionsfrom about 208 to about 726 in the sense strand of FIG. 19 (SEQ IDNO:24); and (4) the complement of any of the DNA molecules of (1)-(3).10. The isolated DNA molecule of claim 9 selected from the groupconsisting of: (1) a DNA molecule which encodes an hIL-1Ra1Lpolypeptide, and which comprises the nucleic acid sequence of nucleotidepositions from about 4 to about 624 in the sense strand of FIG. 15 (SEQID NO:18); (2) a DNA molecule which encodes an hIL-1Ra1S polypeptide,and which comprises the nucleic acid sequence of nucleotide positionsfrom about 4 to about 504 in the sense strand of FIG. 16 (SEQ ID NO:20);(3) a DNA molecule which encodes an hIL-1Ra1V polypeptide, and whichcomprises the nucleic acid sequence of nucleotide positions from about73 to about 726 in the sense strand of FIG. 19 (SEQ ID NO:24); and (4)the complement of any of the DNA molecules of (1)-(3).
 11. The isolatednucleic acid molecule of claim 8 selected from the group consisting of:(1) a DNA molecule comprising the nucleic acid sequence of nucleotidepositions from about 103 to about 681 in the sense strand of FIG. 3 (SEQID NO:6); (2) a DNA molecule comprising the nucleic acid sequence ofnucleotide positions from about 100 to about 465 in the sense strand ofFIG. 7 (SEQ ID NO:12); (3) a DNA molecule comprising the nucleic acidsequence of nucleotide positions from about 244 to about 609 in thesense strand of FIG. 9 (SEQ ID NO:15); and (4) the complement of any ofthe DNA molecules of (1)-(3).
 12. The isolated nucleic acid molecule ofclaim 8 comprising (a) the complete DNA sequence in the sense strand ofFIG. 2 (SEQ ID NO:4), FIG. 3 (SEQ ID NO:6), FIG. 5 (SEQ ID NO:9), FIG. 7(SEQ ID NO:12), or FIG. 9 (SEQ ID NO:15), or (b) the complement of (a).13. An isolated nucleic acid molecule encoding an IL-1lp polypeptide,comprising DNA hybridizing to the complement of a nucleic acid sequenceselected from the group consisting of: (1) the nucleic acid sequenceconsisting of nucleotide positions from about 238 to about 465 in thesense strand of FIG. 7 (SEQ ID NO:12); (2) the nucleic acid sequenceconsisting of nucleotide positions from about 427 to about 609 in thesense strand of FIG. 9 (SEQ ID NO:15); and (3) the nucleic acid sequenceconsisting of nucleotide positions from about 114 to about 135 in thesense strand of FIG. 15 (SEQ ID NO:18).
 14. An isolated nucleic acidmolecule comprising (a) a DNA molecule encoding a polypeptide selectedfrom the group consisting of: (1) a polypeptide comprising the entireamino acid sequence encoded by the longest open reading frame in thecDNA insert in the vector deposited as ATCC Deposit No. 203588; (2) apolypeptide comprising the entire amino acid sequence, or the entireamino acid sequence excluding the 36 N-terminal amino acid residues ofsuch sequence, encoded by the longest open reading frame in the cDNAinsert in the vector deposited as ATCC Deposit No. 203587; (3) apolypeptide comprising the entire amino acid sequence encoded by thelongest open reading frame in the cDNA insert in the vector deposited asATCC Deposit No. 203586; (4) a polypeptide comprising the entire aminoacid sequence, or the entire amino acid sequence excluding theN-terminal amino acid residue of such sequence, encoded by the longestopen reading frame in the cDNA insert in the vector deposited as ATCCDeposit No. 203589; (5) a polypeptide comprising the entire amino acidsequence, or the entire amino acid sequence excluding the N-terminalamino acid residue of such sequence, encoded by the cDNA insert in thevector deposited as ATCC Deposit No. 203590; (6) a polypeptidecomprising the entire amino acid sequence, or the entire amino acidsequence excluding the 34 N-terminal amino acid residues of suchsequence, encoded by the longest open reading frame in the cDNA insertin the vector deposited as ATCC Deposit No. 203846; (7) a polypeptidecomprising the entire amino acid sequence encoded by the longest openreading frame in the cDNA insert in the vector deposited as ATCC DepositNo. 203855; and (8) a polypeptide comprising the entire amino acidsequence, or the entire amino acid sequence excluding the 45 N-terminalamino acid residues of such sequence, encoded by the longest openreading frame in the cDNA insert in the vector deposited as ATCC DepositNo. 203973; or (b) the complement of the DNA molecule of (a).
 15. Anisolated nucleic acid molecule comprising (a) DNA encoding the IL-1lppolypeptide of claim 21, or (b) the complement of the DNA of (a).
 16. Avector comprising the nucleic acid of claim
 1. 17. The vector of claim16 operably linked to control sequences recognized by a host celltransfected with the vector.
 18. A host cell comprising the vector ofclaim
 16. 19. A process for producing an IL-1lp polypeptide comprisingthe steps of: (1) culturing a host cell comprising the DNA molecule ofclaim 15 under conditions suitable for expression of the IL-1lppolypeptide encoded by the DNA molecule; and (2) recovering said IL-1lppolypeptide from the cell culture.
 20. An isolated IL-1lp polypeptideencoded by the nucleic acid molecule of claim
 1. 21. An isolated IL-1lppolypeptide selected from the group consisting of: (1) an hIL-1Ra1Vpolypeptide consisting of an amino acid sequence having at least an 80%sequence identity to the sequence of amino acid residues from about 46to about 218 of FIG. 19 (SEQ ID NO:25); (2) an hIL-1Ra3 polypeptideconsisting of an amino acid sequence having at least an 80% sequenceidentity to the sequence of amino acid residues from about 95 to about134 of FIG. 7 (SEQ ID NO:13); and (3) a mIL-1Ra3 polypeptide consistingof an amino acid sequence having at least an 80% sequence identity tothe sequence of amino acid residues from about 95 to about 134 of FIG. 9(SEQ ID NO:16).
 22. An isolated IL-1lp polypeptide selected from thegroup consisting of: (1) an hIL-1Ra1 polypeptide comprising amino acidresidues from about 37 to about 203 of FIG. 2 (SEQ ID NO:5); (2) anhIL-1Ra1 polypeptide comprising amino acid residues from about 15 toabout 193 of FIG. 3 (SEQ ID NO:7); (3) an hIL-1Ra2 polypeptidecomprising amino acid residues from about 1 to about 134 of FIG. 5 (SEQID NO:10); (4) an hIL-1Ra3 polypeptide comprising amino acid residuesfrom about 95 to about 134 of FIG. 7 (SEQ ID NO:13); (5) a mIL-1Ra3polypeptide comprising amino acid residues from about 95 to about 134 ofFIG. 9 (SEQ ID NO:16); (6) an hIL-1Ra1L polypeptide comprising aminoacid residues from about 26 to about 207 of FIG. 15 (SEQ ID NO:19); (7)an hIL-1Ra1S polypeptide comprising amino acid residues from about 26 toabout 167 of FIG. 16 (SEQ ID NO:21); and (8) an hIL-1Ra1V polypeptidecomprising amino acid residues from about 46 to about 218 of FIG. 19(SEQ ID NO:25).
 23. The isolated IL-1lp polypeptide of claim 22 selectedfrom the group consisting of: (1) an hIL-1Ra1L polypeptide comprisingamino acid residues from about 1 to about 207 of FIG. 15 (SEQ ID NO:19);(2) an hIL-1Ra1S polypeptide comprising amino acid residues from about 1to about 167 of FIG. 16 (SEQ ID NO:21); and (3) an hIL-1Ra1V polypeptidecomprising amino acid residues from about 1 to about 218 of FIG. 19 (SEQID NO:25).
 24. The isolated polypeptide of claim 22 selected from thegroup consisting of: (1) an hIL-1Ra3 polypeptide comprising amino acidresidues from about 34 to about 155 of FIG. 7 (SEQ ID NO:13); and (2) amIL-1Ra3 polypeptide comprising amino acid residues from about 34 toabout 155 of FIG. 9 (SEQ ID NO:16).
 25. The polypeptide encoded by theDNA molecule of (a) in claim
 14. 26. The IL-1lp polypeptide of claim 22that comprises a native amino acid sequence of the IL-1lp fused at itsC-terminus or N-terminus to a heterologous amino acid sequence.
 27. TheIL-1lp polypeptide of claim 26, wherein said heterologous amino acidsequence is an epitope tag sequence.
 28. The IL-1lp polypeptide of claim26, wherein said heterologous amino acid sequence is a Fc region of animmunoglobulin.
 29. An antibody which specifically binds to the IL-1lppolypeptide of claim
 22. 30. The antibody of claim 29, wherein saidantibody is a monoclonal antibody.